Compositions and methods for producing tobacco plants and articles of manufacture with altered alkaloid levels

Abstract

The present disclosure provides tobacco having genetic modifications in a panel of ERF genes. Also provided are tobacco plants having altered total alkaloid and nicotine levels and commercially acceptable leaf grades, the development of the tobacco by breeding or transgenic methods, and the production of tobacco articles from these tobacco plants. Compositions and methods for producing tobacco plants with novel mutations or alleles to reduce nicotine levels are also provided. Also provided are sequence polymorphisms and molecular markers for breeding tobacco having reduced nicotine or alkaloid content while maintaining tobacco leaf grade and tobacco product quality.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 2, 2021, application number 202180057699.3, and invention name “Compositions and methods for producing tobacco plants and products with altered alkaloid levels”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 034,061, filed on June 3, 2020, the entire contents of which are incorporated herein by reference.

[0004] Introduction of sequence listing

[0005] Contains the sequence listing in a file named "P34813WO00_SL.TXT", 2,780,435 bytes ( ), and was created on June 2, 2021, which sequence listing is submitted electronically with this article and is incorporated herein by reference in its entirety. Technical Field

[0006] The present disclosure provides low alkaloid-related chromosome deletion regions (LA-associated regions, LA_associated_regions), tobacco Nic1b loci and genes in or around the regions or loci. Also provided are tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grades, which are developed through breeding or transgenic methods, and tobacco products are produced from these tobacco plants. Background Art

[0007] There are four major alkaloids present in tobacco: nicotine, nornicotine, anabasine, and anatabine. Nicotine is the major alkaloid, typically accounting for more than 90% of the total alkaloids in commercial tobacco cultivars. Nicotine biosynthesis occurs primarily in the tobacco roots. The tobacco plant then transports nicotine through the vascular bundles to the leaves, where it is stored in vacuoles.

[0008] A variety of factors affect tobacco alkaloid levels, including genotype, environment, fertilization and agronomic practices (e.g., nicotine production is stimulated by topping, wounds and herbivore damage). The low alkaloid trait originally found in Cuban cigar tobacco varieties was introduced into cigarette varieties through a series of backcrossing. Low alkaloid tobacco germplasm was subsequently registered in the genetic background of the cultivated variety Burley 21 (Burley 21) (Legg et al., Crop Science, 10: 212 (1970)). Genetic studies using low alkaloid Burley 21 (LA BU21) lines showed that two unconnected loci contribute to the nicotine level in tobacco leaves. These two loci are referred to as Nic1 and Nic2. The nic1 and nic2 (identical to nicotine 1 and nicotine 2, respectively) mutations in LA BU21 are semi-dominant. They show a dose-dependent effect on nicotine levels, with the effect of nic1 being about 2.4 times stronger than that of nic2. Molecular characterization of the Nic2 locus has been reported. The nic2 mutant showed a deletion of a transcription factor gene cluster from the ethylene response factor (ERF) family, such as ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (Shoji et al., Plant Cell, (10): 3390-409 (2010)).

[0009] Reducing the total alkaloid content in tobacco can have many benefits. It can increase the value of tobacco as a biomass resource. The increase of nicotinic alkaloids in tobacco plants can play an important role in protecting plants against insects and herbivores.

[0010] Consistent with the role of alkaloids in insect defense, LA BU21 was reported to be extremely susceptible to insect damage (Legget al., Crop Science, 10:212 (1970)). Further studies comparing flue-cured tobacco isogenic lines with low total alkaloid percentages (approximately 0.20%) to their "normal" duplicate parents (total alkaloids 1.85-2.70%) reported that the low alkaloid lines had lower yield, grade index, total N, and reducing sugar content than the normal flue-cured cultivars (Chaplin and Weeks, Crop Science, 16(3):416-18 (1976)).

[0011] There is a need to identify genes that modulate nicotine levels in tobacco and to develop tobacco plants and products containing altered nicotine levels (eg, reduced nicotine) while maintaining (if not producing improved) leaf quality. Brief description of the invention

[0013] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a genetic modification in a gene and down-regulating the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204 and 205.

[0014] In one aspect, the present disclosure provides a modified tobacco plant or part thereof comprising a genetic modification that targets a gene and downregulates the expression or activity of the gene, wherein the gene encodes a nucleic acid sequence having at least 80% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, and 205.

[0015] In another aspect, the present disclosure provides a modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

[0016] In one aspect, the present disclosure provides a modified tobacco plant or part thereof comprising a recombinant nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule, wherein the non-coding RNA molecule is capable of binding to an mRNA having at least 80% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, and 205.

[0017] In another aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a genetic modification in a gene and down-regulating the expression or activity of the gene, wherein the gene encodes a polypeptide having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0018] In one aspect, the present disclosure provides a modified tobacco plant or part thereof, comprising a genetic modification that targets a gene and downregulates the expression or activity of the gene, wherein the gene encodes a polypeptide having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0019] In one aspect, the present disclosure provides a modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having a nucleic acid sequence encoding a polypeptide having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0020] In another aspect, the present disclosure provides a modified tobacco plant or part thereof comprising a recombinant nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule, wherein the non-coding RNA molecule is capable of binding to an RNA encoding a polypeptide having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207, wherein the non-coding RNA molecule inhibits expression of the polypeptide.

[0021] In one aspect, the present disclosure provides a population of tobacco plants described herein, cured tobacco material from the tobacco plants described herein, and reconstituted tobacco, tobacco blends, and tobacco products made from the cured tobacco material.

[0022] In another aspect, the present disclosure provides a method for producing a tobacco plant with reduced alkaloids, the method comprising: (a) downregulating the expression or activity of a gene encoding (i) a nucleic acid sequence having at least 90% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204, and 205, or (ii) an amino acid sequence having at least 90% identity or similarity to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207; and (b) harvesting leaves or seeds from the tobacco plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Breeding scheme to develop F5 lines with nic1 and nic2 deletion marker alleles (*(x) indicates selfing).

[0024] Figure 2 : Potential alternative splicing variants around locus g31431 and locus g31432. Darker boxes represent detected transcripts, while lighter boxes represent computer-generated CDS models.

[0025] Figure 3: Overexpression of ERF16 but not ERF130, deletion of LA_associated_region in LA BU21 (ie, SEQ ID No. 1, referred to as "Nic1bΔ", which does not contain annotated genes, see Example 2) or g32081 (β-glucosidase 18-like gene), resulted in an increase in nicotine and total alkaloids in LA BU21.

[0026] Figure 4 : Inhibition of individual Nic1b_ERFs (ERFnew, ERF210, ERF199, ERF29 and ERF91L2) via artificial microRNA reduced nicotine levels to approximately 50%-90% of control plants. Mean nicotine levels and standard errors are based on data from Table 7.

[0027] Figure 5A : Quantitative RT-PCR data showing the effect of ERF199-EAR dominant repressor on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Figure 5B :Alkaloid content is altered by expression of the ERF199-EAR dominant repressor.

[0028] Fig. 6A : Quantitative RT-PCR data showing the effect of ERF29-EAR dominant repressor on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Figure 6B : Alkaloid content is altered by expression of the ERF29-EAR dominant repressor.

[0029] Fig. 7A : Quantitative RT-PCR data show the effect of ERF210-EAR dominant repressor on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Figure 7B : Alkaloid content is altered by expression of the ERF210-EAR dominant repressor.

[0030] Fig. 8A : Quantitative RT-PCR data showing the effect of ERF16-EAR dominant repressor on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Figure 8B : Alkaloid content is altered by expression of the ERF16-EAR dominant repressor.

[0031] Fig.9A : Quantitative RT-PCR data showing the effect of ERF130-EAR dominant repressor on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Fig. 9B: Alkaloid content is altered by expression of the ERF130-EAR dominant repressor.

[0032] Fig. 10A : Quantitative RT-PCR data show the effect of ERF91 overexpression ("ERF91 I") or ERF91-EAR dominant suppressor ("ERF91 II") on PMT and QPT gene expression. SPDS gene was used as control. Vector control sample is shown as vc. Fig. 10B : Alkaloid content is altered due to ERF91 overexpression ("ERF91 I") or ERF91-EAR dominant suppressor ("ERF91 II").

[0033] Fig.11A : Quantitative RT-PCR data showing the effect of ERF101 overexpression on PMT and QPT gene expression. SPDS gene was used as a control. The vector control sample is shown as vc. Fig. 11B : Alkaloid content is altered by ERF101 overexpression.

[0034] Fig. 12A : Quantitative RT-PCR data showing the effect of ERFnew overexpression (labeled as ERF17delN) on PMT and QPT gene expression. SPDS gene was used as a control. The vector control sample is shown as vc. Fig. 12B : Alkaloid content was altered by ERFnew overexpression (marked as ERF17delN).

[0035] Fig.13 : Nicotine levels in samples of NIC1b-ERF mutant plants collected from the greenhouse compared to control wild-type TN90. Plants were grown in the 2018 field season and sampled in 2019. MS13646, MS13647, and MS13648 are exemplary mutations of g31420 (NCG1). MS13649, MS13651, and MS13650 are exemplary mutations of ERF16. MS13652 and MS13654 are exemplary mutations of g31435 (NCG15). The dashed lines indicate the highest and lowest points of the wild-type TN90 range. Nicotine levels are expressed as a percentage of dry weight.

[0036] Fig.14: Nornicotine levels in samples of NIC1b-ERF mutant plants collected from the greenhouse compared to control TN90. Plants were grown in the 2018 field season and sampled in 2019. MS13646, MS13647, and MS13648 are exemplary mutations of g31420 (NCG1). MS13649, MS13651, and MS13650 are exemplary mutations of ERF16. MS13652 and MS13654 are exemplary mutations of g31435 (NCG15). The dotted lines indicate the highest and lowest points of the wild-type TN90 range. Nornicotine levels are expressed as a percentage of dry weight.

[0037] Fig.15 : Levels of anabasine in greenhouse collected samples of NIC1b-ERF mutant plants compared to control TN90. Plants were grown in the 2018 field season and sampled in 2019. MS13646, MS13647, and MS13648 are exemplary mutations of g31420 (NCG1). MS13649, MS13651, and MS13650 are exemplary mutations of ERF16. MS13652 and MS13654 are exemplary mutations of g31435 (NCG15). The dashed lines indicate the highest and lowest points of the wild-type TN90 range. Anabasine levels are expressed as a percentage of dry weight.

[0038] Fig.16 : Anatabine levels in greenhouse collected samples of NIC1b-ERF mutant plants compared to control TN90. Plants were grown in the 2018 field season and sampled in 2019. MS13646, MS13647, and MS13648 are exemplary mutations of g31420 (NCG1). MS13649, MS13651, and MS13650 are exemplary mutations of ERF16. MS13652 and MS13654 are exemplary mutations of g31435 (NCG15). The dashed lines indicate the highest and lowest points of the wild-type TN90 range. Anatabine levels are expressed as a percentage of dry weight.

[0039] Fig.17 : Nicotine levels of NIC1b-ERF mutant plant samples collected from the greenhouse compared to the control TN90. Plants were planted in the 2019 field season and sampled in 2020. MS13655, MS13656 and MS13657 are exemplary mutations of g31437 (NCG17). MS13658 and MS13659 are exemplary mutations of ERF130. The dotted lines indicate the highest and lowest points of the wild-type TN 90 range. Nicotine levels are expressed as a percentage of dry weight. The dotted lines indicate the highest and lowest points of the wild-type TN90 range. Nicotine levels are expressed as a percentage of dry weight.

[0040] Fig.18 : Nornicotine levels in NIC1b-ERF mutant plant samples collected from the greenhouse compared to the control TN90. Plants were planted in the 2019 field season and sampled in 2020. MS13655, MS13656 and MS13657 are exemplary mutations of g31437 (NCG17). MS13658 and MS13659 are exemplary mutations of ERF130. The dotted lines indicate the high and low points of the wild-type TN90 range. Nicotine levels are expressed as a percentage of dry weight. The dotted lines indicate the high and low points of the wild-type TN90 range. Nornicotine levels are expressed as a percentage of dry weight.

[0041] Fig.19 : Nicotine levels in greenhouse collected samples of NIC1b-ERF mutant plants compared to control TN90. Plants were planted in the 2019 field season and sampled in 2020. MS13655, MS13656, and MS13657 are exemplary mutations of g31437 (NCG17). MS13658 and MS13659 are exemplary mutations of ERF130. The dashed lines indicate the high and low points of the wild-type TN90 range. Nicotine levels are expressed as a percentage of dry weight. The dashed lines indicate the high and low points of the wild-type TN90 range. Nicotine levels are expressed as a percentage of dry weight.

[0042] Fig. 20 : Anatabine levels of greenhouse collected NIC1b-ERF mutant plant samples compared to control TN90. Plants were grown in the 2019 field season and sampled in 2020. MS13655, MS13656, and MS13657 are exemplary mutations of g31437 (NCG17). MS13658 and MS13659 are exemplary mutations of ERF130. The dashed lines indicate the high and low points of the wild-type TN 90 range. Nicotine levels are expressed as a percentage of dry weight. The dashed lines indicate the high and low points of the wild-type TN90 range. Anatabine levels are expressed as a percentage of dry weight.

[0043] Sequence Description

[0044] SEQ ID No: 1 gives the sequence of LA_associated region identified from TN 90 x LA BU21 hybridization.

[0045] SEQ ID No: 2 gives the sequence of the Nic1b region (including LA_associated_region).

[0046] SEQ ID Nos: 3-37 give the genomic coding sequences (gDNA, which typically start with ATG and end with a stop codon, but in some cases also contain untranslated region (UTR) sequences) of 35 annotated genes (NCGs) in the Nic1b region.

[0047] SEQ ID Nos: 38-72 give the cDNA sequences of 35 annotated genes (NCGs) in the Nic1b region.

[0048] SEQ ID Nos: 73-107 give the amino acid sequences encoded by 35 annotated genes (NCGs) in the Nic1b region.

[0049] Exemplary mature artificial miRNA sequences (sense or antisense) for inhibiting selected NCG genes are given in SEQ ID Nos: 108-119.

[0050] Exemplary guide RNA sequences for editing selected NCG genes are given in SEQ ID Nos: 120-124.

[0051] SEQ ID Nos: 125-145 give 21 SNP marker sequences spanning the Nic1b region or on both sides thereof.

[0052] SEQ ID Nos: 146-148 give the genomic coding, cDNA and amino acid sequences of locus g31432, respectively.

[0053] SEQ ID Nos: 149-151 give the genomic coding, cDNA and amino acid sequences of locus g31446, respectively.

[0054] SEQ ID No: 152-156 gives the genomic coding sequences of various protein coding genes and non-coding RNA from the Nic1b region. SEQ ID No: 157-161 gives the corresponding cDNA sequences. SEQ ID No: 162-183 gives the corresponding protein or non-coding RNA molecular sequences.

[0055] SEQ ID No: 184-186 gives the genomic coding sequences of various transcription factor genes, which show differential expression between normal alkaloid and reduced alkaloid tobacco lines. SEQ ID No: 187-189 gives the corresponding cDNA sequences. SEQ ID No: 190-192 gives the corresponding protein or non-coding RNA molecule sequences.

[0056] SEQ ID Nos: 193-201 give SNP marker sequences that are adjacent to multiple ERF genes or within genes encoding non-coding RNA from the Nic1b region.

[0057] SEQ ID Nos: 202 and 203 give the genomic coding sequences of two ERF genes associated with the Nic1b region, SEQ ID Nos: 204 and 205 give the corresponding cDNA sequences, and SEQ ID Nos: 206 and 207 give the corresponding protein sequences.

[0058] SEQ ID No: 208-214 gives the genomic coding sequences of 7 Nic2_ERF genes, SEQ ID No: 215-221 gives the corresponding cDNA sequences, and SEQ ID No: 222-228 gives the corresponding protein sequences.

[0059] Various sequences include "N" in the nucleotide sequence or "X" in the amino acid sequence. "N" can be any nucleotide, such as A, T, G, C, or a deletion or insertion of one or more nucleotides. In some cases, a string of "N" is given. The number of "N" is not necessarily related to the actual number of undetermined nucleotides at the position. The actual nucleotide sequence may be longer or shorter than the "N" fragment shown. Similarly, "X" can be any amino acid residue or a deletion or insertion of one or more amino acids. Similarly, the number of "X" is not necessarily related to the actual number of undetermined amino acids at the position. The actual amino acid sequence may be longer or shorter than the "X" fragment shown. DETAILED DESCRIPTION OF THE INVENTION

[0061] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art. Those skilled in the art will recognize that many methods can be used in the practice of the present disclosure. In fact, the present disclosure is by no means limited to the methods and materials described. For the purposes of the present disclosure, the following terms are defined as follows.

[0062] Any references cited herein, including, for example, all patents and publications, are incorporated by reference in their entirety.

[0063] As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0064] As used herein, the term "about" means approximately, roughly, roughly, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth.

[0065] As used herein, "Nic1b locus" refers to any chromosomal location or site within or closely associated with the Nic1b region. "Nic1b region" refers to a chromosomal segment of approximately 1.5 million bps in length, corresponding to SEQ ID No. 2 of the TN90 genome, and having alleles associated with the low alkaloid trait. "nic1b mutation" refers to a mutation in the Nic1b locus.

[0066] As used herein, Nic1b_ERF (or plural form, Nic1b_ERFs) refers to any ERF gene or locus at or around the Nic1b locus, and includes, for example, ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See Table 11 and Kajikawa et al., Plant physiol. 2017, 174: 999-1011. "Nic1b_ERF mutation" refers to a mutation in the Nic1b_ERF gene. As used herein, mutations or mutant alleles are shown in all lowercase letters and italics. Gene, locus, or protein names are shown in all uppercase letters or with the first letter capitalized, and can be italicized or non-italicized.

[0067] As used herein, Nic2_ERF (or plural form, Nic2_ERFs) refers to any ERF gene or locus at or around the Nic2 locus, and includes, for example, ERF221, ERF115, ERF168, ERF17, ERF179, ERF189. See Table 12; Shoji et al., Plant Cell, (10): 3390-409 (2010); and Kajikawa et al., Plant physiol. 2017, 174: 999-1011.

[0068] As used herein, mutation refers to heritable genetic modification, which is introduced into a gene to change the expression or activity of a product encoded by the gene. Such modification can be in any sequence region of a gene, such as in a promoter, 5'UTR, exon, intron, 3'UTR or terminator region. In one aspect, mutation reduces, suppresses or eliminates the expression or activity of a gene product. On the other hand, mutation increases, improves, strengthens or increases the expression or activity of a gene product. In one aspect, mutation is not a natural polymorphism present in a specific tobacco variety or cultivar. As used herein, "mutant allele" refers to an allele from a locus, wherein the allele comprises a mutation. As used herein, "mutation" refers to the generation of a mutation that does not involve a transgenic or does not exist in a final mutant that is associated with a mutation in a transgenic. In one aspect, a mutagen is cis. On the other hand, mutagenesis is by gene or genome editing. On the other hand, mutagenesis is by random mutagenesis, such as chemical (such as EMS) or physical (r-irradiation) mutagenesis.

[0069] In one aspect, the mutation is a "non-natural" or "non-naturally occurring" mutation. As used herein, a "non-natural" or "non-naturally occurring" mutation refers to a non-spontaneous mutation produced by human intervention and does not correspond to a spontaneous mutation produced without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted gene modification (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Non-natural mutations and non-naturally occurring mutations do not include naturally occurring spontaneous mutations (e.g., by abnormal DNA replication in plant germ lines).

[0070] Should be understood that, when identifying sudden change, reference dna sequence should be from identical tobacco variety.For example, if the modified tobacco plant comprising sudden change is from TN90 variety, then endogenous reference sequence must be endogenous TN90 sequence, rather than the homologous sequence from different tobacco varieties (for example K326).Similarly, if the modified tobacco cell comprising sudden change is TN90 cell, then endogenous reference sequence must be endogenous TN90 sequence, rather than the homologous sequence from the tobacco cell of different tobacco varieties (for example K326).

[0071] In one aspect, the mutation provided herein produces the dominant allele of the mutation locus. The dominant allele is an allele that masks the contribution of the second allele at the same locus. The dominant allele can be a "dominant negative allele" or a "dominant positive allele". A dominant negative allele or an anti-morph is an allele that is opposite to the normal allele function. The dominant negative allele typically does not work normally and directly inhibits the activity of the wild-type protein (for example, by dimerization) or inhibits the activity of the second protein required for the normal function of the wild-type protein (for example, an activator or downstream component of a pathway). For example, a dominant negative allele eliminates or reduces the normal function of the allele in a heterozygous or homozygous state. A dominant positive allele can increase normal gene function (for example, a hyper-effective allele) or provide a new function (for example, a new allele) for a gene. A semidominant allele occurs when the penetrance of a linked phenotype in individuals heterozygous for the allele is lower than that observed in individuals homozygous for the allele.

[0072] In one aspect, the mutations provided herein produce dominant negative alleles of the mutation locus. In another aspect, the mutations provided herein produce dominant positive alleles of the mutation locus.

[0073] As used herein, "induced" mutation refers to the generation of mutations in polynucleotide sequences by human intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of these methods include the use of chemical mutagens, the use of radiation, and the use of nucleases. In one aspect, induced mutations include the use of a reagent selected from the group consisting of chemical mutagens, radiation, transposons, Agrobacterium, and nucleases.

[0074] In one aspect, inducing mutations comprises using a chemical mutagen. In one aspect, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0075] In another aspect, inducing mutations comprises the use of radiation. In one aspect, the radiation comprises gamma rays, X-rays, or ionizing radiation. In another aspect, the radiation comprises the use of fast neutrons.

[0076] In one aspect, inducing mutations comprises using a transposon. In another aspect, inducing mutations comprises using Agrobacterium.

[0077] In another aspect, inducing mutations comprises the use of a nuclease. In one aspect, the nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, a CRISPR / CasX nuclease, a CRISPR / CasY nuclease, and a Csm1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cas9 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cpf1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasX nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasY nuclease. In one aspect, inducing mutations comprises the use of a Csm1 nuclease.

[0078] In another aspect, inducing mutations comprises the use of a nuclease. In one aspect, the nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, a CRISPR / CasX nuclease, a CRISPR / CasY nuclease, and a Csm1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cas9 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / Cpf1 nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasX nuclease. In one aspect, inducing mutations comprises the use of a CRISPR / CasY nuclease. In one aspect, inducing mutations comprises the use of a Csm1 nuclease.

[0079] When the mutated messenger RNA (mRNA) is translated into a protein or polypeptide, a mutation in a gene coding region (e.g., an exon mutation) can produce a truncated protein or polypeptide. In one aspect, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide comprises at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A comprises 100 amino acids, a truncated form of protein A may comprise 1-99 amino acids.

[0080] In one aspect, the mutations provided herein include null mutations. As used herein, "null mutations" refer to mutations that completely disable the function of a protein encoded by a gene comprising the mutation, or mutations that completely disable the function of a small RNA encoded by a genomic locus. Null mutations can result in a lack of mRNA transcription products, a lack of small RNA transcription products, a lack of protein function, or a combination thereof.

[0081] In one aspect, the mutation in the endogenous gene results in a decrease in expression level compared to an endogenous gene lacking the mutation. In another aspect, the mutation in the endogenous gene results in an increase in expression level compared to an endogenous gene lacking the mutation.

[0082] In one aspect, the non-natural mutation results in a reduced expression level compared to the expression of the gene in a control tobacco plant. In one aspect, the non-natural mutation results in an increased expression level compared to the expression of the gene in a control tobacco plant.

[0083] On the other hand, the mutation in the endogenous gene results in a decrease in the activity level of a protein or polypeptide encoded by the endogenous gene with the mutation compared to a protein or polypeptide encoded by an endogenous gene lacking the mutation. On the other hand, the mutation in the endogenous gene results in an increase in the activity level of a protein or polypeptide encoded by an endogenous gene with the mutation compared to a protein or polypeptide encoded by an endogenous gene lacking the mutation.

[0084] In one aspect, the non-natural mutation results in a protein or polypeptide encoded by a polynucleotide comprising the non-natural mutation having a reduced activity level compared to a protein or polypeptide encoded by a polynucleotide lacking the non-natural mutation. On the other hand, the non-natural mutation results in an increased activity level of a protein or polypeptide encoded by a polynucleotide comprising the non-natural mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-natural mutation.

[0085] In one aspect, the mutation in the genomic locus causes a decrease in expression level compared to a genomic locus lacking a mutation. On the other hand, the mutation in the genomic locus causes an increase in expression level compared to a genomic locus lacking a mutation. On the other hand, the mutation in the genomic locus causes a decrease in the activity level of a protein or polypeptide encoded by a genomic locus with a mutation compared to a protein or polypeptide encoded by a genomic locus lacking a mutation. On the other hand, the mutation in the genomic locus causes an increase in the activity level of a protein or polypeptide encoded by a genomic locus with a mutation compared to a protein or polypeptide encoded by a genomic locus lacking a mutation.

[0086] Gene expression levels are routinely studied in the art. As non-limiting examples, gene expression can be measured using quantitative reverse transcriptase PCR (qRT-PCR), RNA sequencing or Northern blot. In one aspect, qRT-PCR is used to measure gene expression. On the other hand, Northern blot is used to measure gene expression. On the other hand, RNA sequencing is used to measure gene expression.

[0087] As used herein, the tobacco plant can be any plant from the genus Nicotiana, including but not limited to Nicotiana tabacum, Nicotiana amplexicaulis PI 271989; Nicotiana benthamiana PI 555478; Nicotiana bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991; Nicotiana knightiana PI 241013 ... excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana glutinosa PI 241013; Nicotiana glutinosa PI 24101 555527; Nicotiana maritima PI 555535; Nicotiana megalosiphon PI 555536; Nicotiana anudicaulis PI 555540; Nicotiana paniculate PI 555545; Nicotiana aplumbaginifolia PI 555548; Nicotiana repanda PI 555552; ​​Nicotiana rustica; Nicotiana suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana trigonophylla PI 555572. In one aspect, the tobacco plant described herein is a Nicotiana tabacum plant.

[0088] In one aspect, the disclosure provides tobacco plants or parts thereof comprising mutations in the Nic1b_ERF locus, wherein the tobacco plants can produce leaves with a USDA grade index value of 50 or higher when ripening. In one aspect, the tobacco plants also comprise mutations in the ERF gene of the Nic2 locus. In one aspect, the tobacco plants also comprise one or more mutations in two or more, three or more, four or more, five or more, six or more or all seven genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168. In one aspect, the tobacco plants also comprise one or more mutations in ERF189, ERF115 or both. In one aspect, the tobacco plants can produce leaves with a USDA grade index value selected from the group consisting of: 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, and 95 or higher when ripening. On the other hand, tobacco plant can produce the leaf of suitable USDA grade index value when ripening with control plant growing under similar conditions and ripening, wherein said control plant and tobacco plant have basically the same genetic background except sudden change.On the other hand, tobacco plant can produce the leaf of USDA grade index value at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% USDA grade index value when ripening with control plant growing under similar conditions, wherein said control plant and said tobacco plant have basically the same genetic background except sudden change. On the other hand, tobacco plants can produce leaves having a USDA grade index value of 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130% or 120%-130% of the USDA grade index value of the control plant when curing. On the other hand, tobacco plants can produce leaves having a USDA grade index value of 70%-125%, 75%-120%, 80%-115%, 85%-110% or 90%-100% of the USDA grade index value of the control plant when curing. In one aspect, the tobacco plant comprises a nicotine level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growing conditions, wherein the control plant has essentially the same genetic background as the tobacco plant except for the mutation.In another aspect, the tobacco plant comprises a total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. On the other hand, the tobacco plant comprises a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%. On the other hand, the tobacco plant also comprises a transgenic or mutation that directly inhibits the expression or activity of one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP) and MATE transporter.

[0089] In one aspect, the disclosure provides tobacco plants or parts thereof comprising a sudden change in the Nic1b_ERF locus, wherein the tobacco plants can produce leaves having a USDA grade index value comparable to a control plant grown under similar conditions when ripening, wherein the control plant and the tobacco plants have substantially the same genetic background except for the sudden change. In one aspect, the tobacco plants also comprise a sudden change in the ERF gene of the Nic2 locus. In one aspect, the tobacco plants also comprise one or more sudden changes in two or more, three or more, four or more, five or more, six or more or all seven genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168. In one aspect, the tobacco plants also comprise one or more sudden changes in ERF189, ERF115 or both. In another aspect, the tobacco plant can produce leaves having a USDA grade index value selected from the group consisting of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more when cured. In another aspect, the tobacco plant can produce leaves having a USDA grade index value selected from the group consisting of 50-95, 55-95, 60-95, 65-95, 70-95, 75-95, 80-95, 80-95, 85-95, 90-95, 55-90, 60-85, 65-80, 70-75, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, and 90-95 when cured. On the other hand, tobacco plant can produce at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% of the USDA grade index value with the control plant when curing.On the other hand, tobacco plant can produce 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130%, or the leaf of the USDA grade index value of 120%-130% with the USDA grade index value with the control plant when curing. In another aspect, the tobacco plant is capable of producing leaves when cured having a USDA Grade Index value of 70%-125%, 75%-120%, 80%-115%, 85%-110%, or 90%-100% of the USDA Grade Index value of a control plant.On the other hand, the tobacco plant comprises 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the nicotine or total alkaloid level of a control plant grown under similar growth conditions. On the other hand, the tobacco plant also comprises a transgenic or sudden change that directly inhibits the expression or activity of one or more genes, the gene encoding a product selected from the group consisting of PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP) and MATE transporter.

[0090] In one aspect, the Nic1b_ERF locus comprises one or more sequences selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment of less than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 nucleotides. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 nucleotides. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment of 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-3000, 100-4000, 100-5000, 100-6000, 100-7000, 100-8000 or 100-9000 nucleotides. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment of 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, or 8000-9000 nucleotides.

[0091] In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment within 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000 or 70000 nucleotides of a SNP marker selected from the group consisting of SEQ ID Nos. 125-145 and 193-201. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment within 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-3000, 100-4000, 100-5000, 100-6000, 100-7000, 100-8000 or 100-9000 nucleotides of a SNP marker selected from the group consisting of SEQ ID Nos. 125-145 and 193-201. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment within 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000 or 8000-9000 nucleotides of a SNP marker selected from the group consisting of SEQ ID Nos. 125-145 and 193-201.

[0092] In one aspect, the Niclb_ERF locus comprises a sequence selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment within 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000 or 70000 nucleotides selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In another aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment of 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-3000, 100-4000, 100-5000, 100-6000, 100-7000, 100-8000, 100-9000 nucleotides selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment within 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000 nucleotides selected from the group consisting of: SEQ ID Nos. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205.

[0093] In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment flanked by and not comprising any two sequences selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment flanked by and not comprising any two of the SNP markers selected from the group consisting of SEQ ID No. 125-145 and 193-201. In one aspect, the Nic1b_ERF locus comprises a sequence or chromosome segment flanked by and not comprising any two sequences selected from the group consisting of SEQ ID No. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205.

[0094] In one aspect, the present disclosure also provides a tobacco variety, cultivar or line comprising a mutation selected from the group consisting of a nic1b_erf mutation, a nic2 mutation and a combination thereof, wherein the tobacco variety, cultivar or line has a leaf grade comparable to the leaf grade of a control tobacco variety, cultivar or line grown under similar growing conditions, wherein the control tobacco variety has substantially the same genetic background as the tobacco variety, cultivar or line except for the mutation.

[0095] In one aspect, the disclosure also provides a non-transgenic tobacco plant or part thereof comprising a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% and less than 0.05%, wherein the tobacco plant is capable of producing leaves having a USDA grade index value of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more and 95 or more when cured. In another aspect, such a non-transgenic tobacco plant comprises a nicotine level of less than 2.0% and is capable of producing leaves having a USDA grade index value of 70 or more when cured. In another aspect, such non-transgenic tobacco plants comprise nicotine levels of less than 1.0% and, when cured, are capable of producing leaves having a USDA Grade Index value of 70 or greater.

[0096] In one aspect, the present disclosure also provides a tobacco plant or part thereof comprising a non-transgenic mutation, wherein the non-transgenic mutation reduces the nicotine or total alkaloid level of the tobacco plant to less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level of a control plant grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value upon curing that is comparable to the USDA Grade Index value of the control plant, and wherein the control plant has essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

[0097] In one aspect, the disclosure provides tobacco plants or parts thereof comprising mutations in the Nic1b_ERF locus, wherein the mutations are not present in LA Burley 21. In one aspect, the tobacco plants provided herein comprise shorter chromosome introgressions in the Nic1b_ERF locus compared to LA Burley 21. In another aspect, the tobacco plants provided herein do not comprise the deletion of a complete gene or a complete gene coding sequence in the Nic1b_ERF locus. In one aspect, the tobacco plants provided herein are homozygous in the Nic1b_ERF locus. In another aspect, the tobacco plants provided herein are heterozygous in the Nic1b_ERF locus. In one aspect, the tobacco plants provided herein comprise a Nic1b_ERF mutation selected from the group consisting of a point mutation, a deletion, an insertion, a duplication, and an inversion. In one aspect, the Nic1b_ERF mutation in the tobacco plants provided herein is introduced by a pathway selected from the group consisting of random mutagenesis and targeted mutagenesis. In another aspect, the Nic1b_ERF mutation of the tobacco plant provided herein is introduced by a targeted mutagenesis method selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALEN, and a CRISPR.

[0098] In one aspect, the tobacco plant provided herein comprises one or more mutations in one or more genes, the gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 153, 154, 202, 203 and 208-214, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 153, 154, 202, 203 and 208-214, and fragments thereof.

[0099] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52 to 54, 158, 159, 204, 205 and 215-221, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 38, 48, 49, 52-54, 158, 159, 204, 205 and 215-221, and fragments thereof.

[0100] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207, and 222-228, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207, and 222-228, and fragments thereof.

[0101] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof.

[0102] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205 and 54, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes comprising a coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205 and 54, and fragments thereof.

[0103] In one aspect, the tobacco plants provided herein comprise one or more mutations in one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88 and 89, and fragments thereof. In one aspect, the one or more mutations reduce the expression or activity of one or more genes encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88 and 89, and fragments thereof.

[0104] LA Burley 21 (also known as LA BU21) is a low total alkaloid tobacco line produced by introducing a low alkaloid gene from a Cuban cigar variety into Burley 21 through several backcrosses (Legg et al., 1970). It has about 0.2% total alkaloids (dry weight) compared to about 3.5% (dry weight) for its parent Burley 21. The leaf grade of LA BU21 is well below commercially acceptable standards. LA BU21 also exhibits other unfavorable leaf phenotypes characterized by low yield, delayed maturity and senescence, higher susceptibility to insect herbivory, and poor final product quality after ripening (Chaplin and Weeks, Crop Sci. 16: 416-418 (1976); Legg et al. Crop. Sci. 10: 212 (1970); Chaplin and Burk, Crop Sci. 75: 133-136 (1983)). LA BU21 leaves also exhibited traits such as higher polyamine content, higher chlorophyll content and more mesophyll cells per unit leaf area.

[0105] Unless otherwise stated, the measured value of alkaloid, polyamine or nicotine level (or other leaf chemistry or characteristic characterization) or leaf grade index value of the tobacco plant, kind, cultivar or strain mentioned herein refers to average measured value, comprises, for example the mean value of a plurality of leaves of single representative plant, or the average measured value of the representative colony of the tobacco plant from single kind, cultivar or strain.Unless otherwise stated, after topping for two weeks, in the leaf sample collected by the 3rd, 4th and 5th leaf after topping, measure the nicotine, alkaloid or polyamine level (or other chemistry or characteristic characterization) of tobacco plant described herein.On the other hand, after topping, in the leaf with the highest nicotine, alkaloid or polyamine level (or other leaf chemistry or characteristic characterization), measure the nicotine, alkaloid or polyamine level (or other leaf chemistry or characteristic characterization) of tobacco plant. In one aspect, the nicotine, alkaloid or polyamine levels of the tobacco plant are measured after topping in the following leaf numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In another aspect, the nicotine, alkaloid or polyamine levels (or other leaf chemistry or characteristic) of the tobacco plant are measured after topping in two or more leaves with consecutive leaf numbers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30. On the other hand, the nicotine, alkaloid or polyamine level (or other leaf chemistry or characteristic characterization) of tobacco plant is measured in leaves after topping, and the leaf numbering is selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. On the other hand, the nicotine, alkaloid or polyamine level (or other leaf chemistry or characteristic characterization) of tobacco plant is measured in two or more leaves after topping, and the leaf numbering is selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30. On the other hand, the nicotine, alkaloid or polyamine level (or other leaf chemistry or characteristic characterization) of tobacco plant is measured in three or more leaves after topping, and the leaf numbering is selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30.

[0106] As used herein, leaf numbering is based on the leaf position on the tobacco stem, with leaf number 1 being the newest leaf after topping (top) and the highest leaf number being assigned to the oldest leaf (bottom).

[0107] The population of tobacco plants or collection of leaves used to determine the average measurement (e.g., alkaloid or nicotine level or leaf grade) can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-accepted standards are followed to determine the average measurement or grade index value.

[0108] As used herein, " topping " refers to when tobacco plant approaches vegetative maturity and when reproductive growth begins, removes the stem tip, comprises SAM, flower and several adjacent leaves at the most.Typically, tobacco plant topping is carried out in the bud stage (soon after the flower begins to appear).For example, when 50% of the plant had at least one open flower, the tobacco plant of greenhouse or field growth can be toppled.Topping tobacco plant makes the loss of apical dominance and also induces alkaloid output to increase.

[0109] Typically, about 2 weeks after topping, the nicotine, alkaloid or polyamine levels (or other leaf chemistry or characteristic characterization) of tobacco plant are measured.Also other time points are available.In one aspect, about 1,2,3,4 or 5 weeks after topping, the nicotine, alkaloid or polyamine levels (or other leaf chemistry or characteristic characterization) of tobacco plant are measured.On the other hand, about 3,5,7,10,12,14,17,19 or 21 days after topping, the nicotine, alkaloid or polyamine levels (or another leaf chemistry or characteristic characterization) of tobacco plant are measured.

[0110] As used herein, "similar growing conditions" or "comparable growing conditions" refers to similar environmental conditions and / or agronomic practices used to grow two or more plant genotypes and to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorus). Agronomic practices include, for example, sowing, pruning, undercutting, transplanting, topping, and suctioning. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103.

[0111] "Alkaloids" are complex nitrogen-containing compounds that occur naturally in plants and have pharmacological effects in humans and animals. "Nicotine" is the major natural alkaloid in commercial cigarette tobacco, accounting for about 90% of the alkaloid content in tobacco. Other major alkaloids in tobacco include cotinine, nornicotine, myosamine, nicotine, anabasine, and anatabine. Minor tobacco alkaloids include nicotine-n-oxide, N-methyl anatabine, N-methyl anabasine, pseudodeoxynicotine, 2,3-bipyridine, and others.

[0112] In one aspect, compared with the control tobacco plant of the transgenic that does not have nic1b_erf sudden change or Nic1b_ERF guide when growing under similar growth conditions, tobacco plant provided herein comprises lower level of total alkaloid or independent alkaloid.On the other hand, compared with the control tobacco plant of growing under similar growth conditions, tobacco plant provided herein comprises lower level of one or more alkaloids that are selected from lower group: cotinine, nornicotine, mysmine, nicotine, anabasine and anatabine.In one aspect, lower alkaloid or nicotine level refer to the alkaloid of control tobacco plant or nicotine level 1% below, 2% below, 5% below, 8% below, 10% below, 12% below, 15% below, 20% below, 25% below, 30% below, 40% below, 50% below, 60% below, 70% below or 80% below alkaloid or nicotine level. In another aspect, lower alkaloid or nicotine levels refer to alkaloid or nicotine levels that are about 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 11%-12%, 12%-13%, 13%-14%, 14%-15%, 15%-16%, 16%-17%, 17%-18%, 18%-19%, 19%-20%, 21%-22%, 22%-23%, 23%-24%, 24%-25%, 25%-26%, 26%-27%, 27%-28%, 28%-29%, or 29%-30% of the alkaloid or nicotine level of a control tobacco plant. In another aspect, lower alkaloid or nicotine levels refer to alkaloid or nicotine levels that are about 0.5%-5%, 5%-10%, 10%-20%, 20%-30% of the alkaloid or nicotine level of a control tobacco plant.

[0113] Alkaloid levels can be determined by methods known in the art, such as by quantification based on gas-liquid chromatography, high performance liquid chromatography, radioimmunoassay and enzyme-linked immunosorbent assay. For example, nicotinic alkaloid levels can be determined by methods based on CORESTA Recommended Method No. 7, 1987 and ISO standards (ISO TC 126N 394 E. For methods using gas-liquid chromatography equipped with a capillary column and a FID detector, see also Hibi et al., Plant Physiology 100: 826-35 (1992)). Unless otherwise stated, all alkaloid levels described herein are measured using those methods defined in CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55 on March 23, 1999 (and Vol. 74, No. 4, revised on January 7, 2009).

[0114] Alternatively, tobacco total alkaloids can be measured using segmented flow colorimetry, which is improved by Skalar Instrument Co (West Chester, PA) and described in Collins et al., Tobacco Science 13: 79-81 (1969) for analyzing tobacco samples. In brief, before analyzing total alkaloids and reducing sugars, tobacco samples are dried, ground and extracted. Then, the method uses acetic acid / methanol / water extraction and charcoal to decolorize. The determination of total alkaloids is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex, which is measured at 460nm. Unless otherwise stated, total alkaloid levels or nicotine levels described herein are based on dry weight (e.g., total alkaloid percentage or nicotine percentage).

[0115] In one aspect, the tobacco plants provided herein comprise lower levels of nicotine compared to transgenic control tobacco plants without the Nic1b_ERF mutation or Nic1b_ERF guidance when grown under similar growth conditions. In one aspect, lower nicotine levels refer to an average nicotine level of less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the average nicotine level of the control tobacco plants. In another aspect, lower nicotine levels refer to an average nicotine level of about 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 11%-12%, 12%-13%, 13%-14%, 14%-15%, 15%-16%, 16%-17%, 17%-18%, 18%-19%, 19%-20%, 21%-22%, 22%-23%, 23%-24%, 24%-25%, 25%-26%, 26%-27%, 27%-28%, 28%-29%, or 29%-30% of the average nicotine level of control tobacco plants. In another aspect, lower nicotine levels refer to average nicotine levels of about 0.5%-5%, 5%-10%, 10%-20%, 20%-30% of the average nicotine level of control tobacco plants.

[0116] In one aspect, the tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5. %, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8% and 9%. In another aspect, the tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%-0.02%, 0.02%-0.05%, 0.05%-0.75%, 0.75%-0.1%, 0.1%-0.15%, 0.15%-0.2%, 0.2%-0.3%, 0.3%-0.35%, 0.35%-0.4%, 0.4%-0.5%, 0.5%-0.6%, 0.6%-0.7%, 0.7%-0.8%, 0.8%-0.9%, 0.9%-1%, 1%-1.1%, 1.1%-1.2%, 1.2%-1.3% on a dry weight basis. , 1.3%-1.4%, 1.4%-1.5%, 1.5%-1.6%, 1.6%-1.7%, 1.7%-1.8%, 1.8%-1.9%, 1.9%-2%, 2%-2.1%, 2.1%-2.2%, 2.2%-2.3%, 2.3%-2.4%, 2.4%-2.5%, 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, 2.9%-3%, 3%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, 3.4%-3.5%, and 3.5%-3.6%. In another aspect, the tobacco plants provided herein comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%-0.1%, 0.02%-0.2%, 0.03%-0.3%, 0.04%-0.4%, 0.05%-0.5%, 0.75%-1%, 0.1%-1.5%, 0.15%-2%, 0.2%-3% and 0.3%-3.5% by dry weight.

[0117] The disclosure also provides the nicotine level with change and other tobacco characters (for example leaf grade index value) do not have the tobacco plant of negative effect.In one aspect, low nicotine or nicotine-free tobacco variety provides the cured tobacco of commercially acceptable grade.Based on the following factors, assess tobacco grade, include but not limited to petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (with the tinting intensity and depth and gloss of leaf relevant), hygroscopicity (tobacco leaf absorbs and retains the ability of environmental moisture) and green nuance or come off.Leaf grade can, for example, use the official standard grade announced by the Agricultural Marketing Service (7U.SC § 511) of the USDepartment of Agriculture to determine. See, e.g., the official standard grades of Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55 F.R. 40645); the official standard grades of Flue-cured Tobacco (U.S. Type 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54 F.R. 7925); the official standard grades of Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 F.R. 16854); the official standard grades of Ohio Cigar Tobacco (U.S. Type 42, 43 and 44), effective December 8, 1963 (28 F.R. 1171928 and 28 F.R. 11926); the official standard grades of Wisconsin Cigar Tobacco (U.S. Type 41 ... March 27, 1989 (54 F.R. 7925); the official standard grades of Wisconsin Cigar Tobacco (U.S. Type 41, 43 and 44), effective March 27, 1989 (54 F.R. 7925); the official standard grades of Wisconsin Ci Official Standard Grades for Cigar-Binder Tobacco (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), effective April 1971. USDA grade index values ​​may be determined based on industry-accepted grade indices. See, e.g., Bowman et al, Tobacco Science, 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the above references are incorporated by reference in their entirety).Unless otherwise indicated, the USDA grade index for any plant described herein is a 0-100 numerical representation of the received federal grade and is a weighted average of all stem positions. A higher grade index indicates higher quality. Alternatively, leaf grade can be determined by hyperspectral imaging. See, for example, WO 2011 / 027315 (published on March 10, 2011, and the entire contents of which are incorporated by reference).

[0118] In one aspect, compared to control tobacco plants grown under similar growth conditions, the tobacco plants provided herein comprise similar levels of one or more tobacco aromatic compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoid, cembrenoid, sugar esters and reducing sugars. On the other hand, the tobacco plants provided herein comprise nic1b_erf mutations, nic2 mutations or a combination thereof, which have no effect on the levels of one or more tobacco aromatic compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoid, cembrenoid, sugar esters and reducing sugars.

[0119] As used herein, tobacco aroma compounds are compounds relevant to the local flavor and fragrance of tobacco smoke.These compounds include but are not limited to 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoid and labdenoid diterpenes and sugar esters.The concentration of tobacco aroma compounds can be measured by any known metabolite pattern method in the art, including but not limited to gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry.Referring to The Handbook of Plant Metabolomics edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

[0120] As used herein, "reducing sugar" is any sugar (monosaccharide or polysaccharide) with free or potentially free aldehyde or ketone groups. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing smoke pH and effectively reducing the amount of "free" unprotonated nicotine. For example, reducing sugars balance the taste of tobacco by changing the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content in different tobacco varieties, the same variety, and the same plant strain caused by growing conditions has been reported. Reducing sugar levels can be measured using segmented flow colorimetry, which is described by Davis, Tobacco Science 20: 139-144 (1976) and improved by Skalar Instrument Co (West Chester, PA) for analyzing tobacco samples. For example, the sample is dialyzed with a sodium carbonate solution. The new copper reagent is added to the sample and the solution is heated. The new copper reagent chelate is reduced in the presence of sugar to obtain a colored complex measured at 460nm.

[0121] In one aspect, the tobacco plants provided herein comprise one or more non-naturally occurring mutant alleles at the Nic1b_ERF or Nic2 loci that reduce or eliminate one or more gene activities from the Nic1b_ERF or Nic2 loci. In one aspect, these mutant alleles result in lower nicotine levels. Mutant Nic1b_ERF or Nic2 alleles can be introduced by any method known in the art, including random or targeted mutagenesis approaches.

[0122] Such mutagenesis methods include, but are not limited to, treatment of seeds with ethyl methyl sulfate (EMS) (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon press, pp317-320, 1965) or UV-irradiation, X-rays, and fast neutron radiation (see, e.g., Verkerk, Neth. J. Agric. Sci. 19: 197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup.rd ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. No. 4,732,856 and U.S. Pat. No. 5,013,658), and T-DNA insertion methods (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists of chemically induced random point mutations over the length of the genome. Fast neutron mutagenesis comprises that seed is exposed to neutron bombardment, and it causes large disappearance by double-stranded DNA break.Transposon marker is included in the expression of inserting transposon to reduce or eliminate gene in endogenous gene.The type of mutation that may exist in tobacco gene comprises for example point mutation, disappearance, insertion, duplication and inversion.This type of sudden change is ideally present in the coding region of tobacco gene; Yet, the sudden change in the promoter region of tobacco gene and intron or the non-translational region also may be required.

[0123] In addition, a rapid and automatable method of screening chemically induced mutations TILLING (targeted local lesions induced in the genome) using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the present disclosure. See, McCallum et al. (2000) Nat. Biotechnol. 18: 455-457. Mutations that affect gene expression or interfere with gene function can be determined using methods well known in the art. Insertion mutations in gene exons typically result in null mutants. Mutations in conserved residues can be particularly effective in inhibiting protein function. In one aspect, a tobacco plant comprising a nonsense mutation (e.g., a stop codon) is one or more NCG genes described herein.

[0124] In one aspect, the disclosure also provides tobacco strains with changed nicotine levels while maintaining commercially acceptable leaf quality. These strains can be produced by introducing mutations into one or more genes at Nic1b_ERF or Nic2 loci via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly spaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, e.g., U.S. Patent Application Publication 2017 / 0233756). See, e.g., Gaj et al., Trends in Biotechnology, 31 (7): 397-405 (2013).

[0125] The screening and selection of mutagenic tobacco plants can be by any method known to those of ordinary skill in the art. The example of screening and selection method includes but is not limited to Southern analysis, the pcr amplification for detecting polynucleotide, Northern blot, RNA enzyme protection, primer extension, the RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next generation sequencing technology (for example Illumina, PacBio, Ion Torrent, 454), the enzyme assay for detecting the enzyme or ribozyme activity of polypeptide and polynucleotide, and protein gel electrophoresis, Western blot, immunoprecipitation and enzyme-linked immunosorbent assay for detecting polypeptide. Other techniques (such as in situ hybridization, enzyme staining and immunostaining) also can be used for detecting the presence or expression of polypeptide and / or polynucleotide. It is known to be used to perform the method for all reference techniques.

[0126] In one aspect, the tobacco plant or plant genome provided herein is mutated or edited by a nuclease selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 nuclease, or a CRISPR / Csm1 nuclease.

[0127] As used herein, "editing" or "genome editing" refers to targeted mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an endogenous plant genomic nucleic acid sequence, or the removal or replacement of an endogenous plant genomic nucleic acid sequence. In one aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to the endogenous nucleic acid sequence. In one aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80% or at least 75% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, 202-205 and 208-221, and fragments thereof. In another aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80% or at least 75% sequence identity to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, 202-205 and 208-221.

[0128] Meganucleases, ZFNs, TALENs, CRISPR / Cas9, CRISPR / Csm1, and CRISPR / Cpf1 induce double-stranded DNA breaks at target sites in genomic sequences, which are then repaired by the natural process of homologous recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the cleavage site, which may include deletions or insertions that lead to gene disruption in the case of NHEJ, or integration of donor nucleic acid sequences by HR. In one aspect, the method provided includes editing a plant genome with a nuclease, and the nuclease provided mutates at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 nucleotides in the plant genome by HR with a donor polynucleotide. In one aspect, the mutation provided is caused by genome editing using a nuclease. On the other hand, the mutation provided is caused by non-homologous end joining or homologous recombination.

[0129] In one aspect, the mutations provided herein provide dominant mutants that activate the expression or activity of a target gene, such as a gene selected from the group consisting of a biosynthetic enzyme, a regulatory transcription factor, a transporter, a catabolic enzyme, or a combination thereof, for use as one or more antioxidants.

[0130] Meganucleases, usually identified in microorganisms, are unique enzymes with high activity and long recognition sequences (>14 bp), allowing site-specific digestion of target DNA. Engineered forms of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14-40 bp). The engineering of meganucleases may be more challenging than the engineering of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Dedicated methods of mutagenesis and high-throughput screening have been used to generate new meganuclease variants that recognize unique sequences and have improved nuclease activity.

[0131] ZFNs are synthetic proteins consisting of an engineered zinc finger DNA binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be designed to cleave almost any long double-stranded DNA to modify the zinc finger DNA binding domain. ZFNs form dimers from monomers consisting of the nonspecific DNA cleavage domain of the FokI endonuclease fused to a zinc finger array that is engineered to bind to a target DNA sequence.

[0132] The DNA binding domain of ZFN is typically composed of 3-4 zinc finger arrays. The amino acids at positions -1, +2, +3 and +6 relative to the start of the zinc finger ∞-helix (which contribute to site-specific binding to the target DNA) can be changed and customized to fit the specific target sequence. Other amino acids form a common backbone to produce ZFNs with different sequence specificities. The rules for selecting the target sequence of ZFN are known in the art.

[0133] The FokI nuclease domain requires dimerization to cut DNA, and therefore requires two ZFNs with their C-terminal regions to bind to the opposite DNA strands of the cutting site (5-7 bp apart). If the two ZF binding sites are palindromic, the ZFN monomer can cut the target site. As used herein, the term ZFN is broad and includes monomeric ZFNs that can cut double-stranded DNA without the help of another ZFN. The term ZFN is also used to refer to one or two elements of a pair of ZFNs that are engineered to work together to cut DNA at the same site.

[0134] Without being limited by any scientific theory, since the DNA binding specificity of zinc finger domains can in principle be re-engineered using one of a variety of methods, customized ZFNs can theoretically be constructed to target almost any gene sequence. Publicly available methods for engineering zinc finger domains include neighbor assembly (CoDA), oligomer engineering (OPEN), and modular assembly (Modular Assembly).

[0135] TALEN is an artificial restriction enzyme produced by fusing the transcription activator-like effector (TALE) DNA binding domain with the FokI nuclease domain. When each member of the TALEN pair binds to the DNA sites on both sides of the target site, the FokI monomer dimerizes and causes double-stranded DNA breaks at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cut double-stranded DNA without the help of another TALEN. The term TALEN is also used to refer to one or two elements of a pair of TALENs that work together to cut DNA at the same site.

[0136] Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any DNA sequence. TALE proteins are DNA binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. Xanthomonas pathogens secrete TALEs into host plant cells during infection. TALEs move to the nucleus, where they recognize and bind to specific DNA sequences in the promoter region of specific genes in the host genome. TALEs have a central DNA binding domain consisting of 13-28 repeating monomers of 33-34 amino acids. Except for the highly variable amino acid residues at positions 12 and 13, the amino acids of each monomer are highly conserved. These two variable amino acids are referred to as repeat variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and regulation of RVDs can recognize continuous DNA bases. This simple relationship between amino acid sequence and DNA recognition allows the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing the appropriate RVD.

[0137] In addition to the wild-type FokI cleavage domain, variants with mutated FokI cleavage domains have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for targeting sites in the genome with appropriate orientation and spacing. The number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between two separate TALEN binding sites are parameters for achieving high levels of activity.

[0138] The relationship between the amino acid sequence of the TALE binding domain and the DNA recognition allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., .Nucleic Acids Research (2012) 40: W117-122.; Cermak et al., Nucleic Acids Research (2011). 39: e82; and tale-nt.cac.cornell.edu / about.

[0139] The CRISPR / Cas9 system, CRISPR / Csm1 or CRISPR / Cpf1 system is an alternative to the FokI-based methods ZFN and TALEN. The CRISPR system is based on RNA-guided engineered nucleases that use complementary base pairing to recognize DNA sequences at the target site.

[0140] CRISPR / Cas9, CRISPR / Csm1 and CRISPR / Cpf1 systems are part of the adaptive immune system of bacteria and archaea, protecting them from invasion of invasive nucleic acids (such as viruses) by cutting foreign DNA in a sequence-dependent manner. Immunity is obtained by integrating a short fragment of invasive DNA called a spacer between two adjacent repeats at the proximal end of the CRISPR locus. The CRISPR array (including the spacer) is transcribed when it encounters invasive DNA later and processed into a small interfering CRISPR RNA (crRNA) of about 40 nt in length, which is combined with a trans-activating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. This cuts a homologous double-stranded DNA sequence called a protospacer in the invasive DNA. A prerequisite for cutting is the presence of a conserved protospacer adjacent motif (PAM) downstream of the target DNA, which typically has a sequence of 5-NGG-3, but a low frequency of NAG. Specificity is provided by a so-called "seed sequence" of about 12 bases upstream of the PAM, which must match between RNA and target DNA. Cpf1 and Csm1 act in a manner similar to Cas9, but Cpf1 and Csm1 do not require tracrRNA.

[0141] In another aspect, the tobacco plants provided further comprise one or more mutations in one or more loci encoding nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10), which confer reduced amounts of nornicotine compared to control plants lacking one or more mutations in one or more loci encoding nicotine demethylase (see U.S. Patent Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706). In one aspect, the modified tobacco plants described further comprise reduced nicotine demethylase activity compared to control plants grown and matured under comparable conditions.

[0142] The present disclosure also provides compositions and methods for inhibiting the expression or function of one or more polypeptides from the Nic1b_ERF locus in a plant, particularly a Nicotiana plant, including various commercial varieties of tobacco plants.

[0143] In one aspect, the present disclosure provides a tobacco plant or part thereof comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of: SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and wherein the inhibitory sequence is linked to at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides have at least 90% sequence identity, and the sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof.In another aspect, the present disclosure provides a tobacco plant or part thereof comprising a heterologous expression cassette comprising a Nic1b_ERF inhibitory sequence of a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of: SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and wherein the inhibitory sequence is linked to at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides have at least 90% identity, and the sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205, and fragments thereof. In one aspect, the Nic1b_ERF inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of a single-stranded RNA polynucleotide, a double-stranded RNA polynucleotide, and a combination thereof. In one aspect, the Nic1b_ERF inhibitory sequence comprises a sequence selected from the group consisting of SEQ ID No. 108-119.

[0144] As used herein, the term "inhibit / inhibition / inhibiting" is defined as any method known in the art or described herein that reduces the expression or function of a target gene product (e.g., a target gene product). "Inhibition" can be performed in the context of a comparison between two plants, such as a genetically modified plant vs. a wild-type plant. Alternatively, the inhibition of the expression or function of a target gene product can be performed in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between different plants, and includes comparisons between developmental stages or time stages within the same plant or plant part, or between plants or plant parts. "Inhibition" includes any relative reduction in the function or production of a target gene product, up to and including complete elimination of the function or production of the gene product. The term "inhibition" encompasses any method or composition that downregulates the translation and / or transcription of a target gene product or the functional activity of a target gene product. In one aspect, the mRNA or protein level of one or more genes of the Nic1b locus in the modified plant is 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% less than the mRNA or protein level of the same gene in a non-mutant or a plant that has not been genetically modified to inhibit the expression of the gene.

[0145] The term "inhibitory sequence" encompasses any polynucleotide or polypeptide sequence capable of inhibiting the expression or function of a gene involved in the regulation of nicotine biosynthesis at the Nic1b_ERF locus in a plant, such as a full-length polynucleotide or polypeptide sequence, a truncated polynucleotide or polypeptide sequence, a fragment of a polynucleotide or polypeptide sequence, a variant of a polynucleotide or polypeptide sequence, a sense-oriented nucleotide sequence, an antisense-oriented nucleotide sequence, a complement of a sense or antisense-oriented nucleotide sequence, an inverted region of a nucleotide sequence, a hairpin of a nucleotide sequence, a double-stranded nucleotide sequence, a single-stranded nucleotide sequence, a combination thereof, etc. The term "polynucleotide sequence" includes sequences of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, a combination thereof, etc.

[0146] The inhibitory sequence is specified by the name of the target gene product. Therefore, a "Nic1b_ERF inhibitory sequence" refers to an inhibitory sequence that is capable of inhibiting the expression of a gene involved in the regulation of nicotine biosynthesis (e.g., at the transcription and / or translation level) of the Nic1b_ERF locus in a plant, or is capable of inhibiting the function of a gene product. When the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, it means that the inhibitory sequence itself exerts an inhibitory effect; or, when the inhibitory sequence encodes an inhibitory nucleotide molecule (e.g., a hairpin RNA, miRNA, or double-stranded RNA polynucleotide), or encodes an inhibitory polypeptide (e.g., a polypeptide that inhibits the expression or function of a target gene product), after its transcription (e.g., in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the product is transcribed or translated, respectively, and exerts an inhibitory effect on the target gene product (e.g., inhibiting the expression or function of the target gene product).

[0147] The disclosed Nic1b_ERF inhibitory sequences can be sequences that trigger gene silencing via any silencing pathway or mechanism known in the art, including but not limited to sense suppression / co-suppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference, ribozymes, small interfering RNA, artificial or synthetic microRNA, and artificial trans-acting siRNA. Depending on the desired results, the Nic1b_ERF inhibitory sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, and up to the full-length polynucleotide encoding the protein of the present disclosure. In one aspect, the Nic1b_ERF inhibitory sequence can be a fragment of about 50 to about 400 nucleotides, about 70 to about 350 nucleotides, about 90 to about 325 nucleotides, about 90 to about 300 nucleotides, about 90 to about 275 nucleotides, about 100 to about 400 nucleotides, about 100 to about 350 nucleotides, about 100 to about 325 nucleotides, about 100 to about 300 nucleotides, about 125 to about 300 nucleotides, or about 125 to about 275 nucleotides in length. In some embodiments, the fragment length of the cytochrome P450 polynucleotide is about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 400 nucleotides, and other such values ​​between about 70 and about 400 nucleotides. In one aspect, the Niclb_ERF inhibitory sequence can comprise about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0148] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that polynucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.

[0149] In one aspect, the disclosure provides a recombinant DNA construct comprising a promoter, the promoter is functional in tobacco cells and is operably linked to a polynucleotide encoding an RNA molecule, the RNA molecule is capable of binding to an RNA encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 83, 84, 87-89, 180, 181, 206 and 207, and 222-228, and fragments thereof, and wherein the RNA molecule inhibits the expression of the polypeptide. In one aspect, the RNA molecule is selected from the group consisting of microRNA, siRNA, and trans-acting siRNA. In another aspect, the recombinant DNA construct encodes a double-stranded RNA. Transgenic tobacco plants or parts thereof, cured tobacco materials, or tobacco products comprising these recombinant DNA constructs are also provided. In one aspect, these transgenic plants, cured tobacco materials, or tobacco products contain lower levels of nicotine compared to control tobacco plants not containing the recombinant DNA construct. Further provided is a method of reducing nicotine levels in a tobacco plant comprising transforming the tobacco plant with any of these recombinant DNA constructs.

[0150] As used herein, "operably linked" refers to a functional connection between two or more elements. For example, an operable connection between a target polynucleotide and a regulatory sequence (e.g., a promoter) is a functional connection that allows the target polynucleotide to be expressed. Operably linked elements can be continuous or non-continuous.

[0151] As used herein, and when used to refer to a sequence, "heterologous" refers to a sequence derived from an alien species, or if from the same species, a sequence substantially modified from its native form in composition and / or genomic locus by intentional human intervention. The term is also applicable to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs". In this way, "heterologous" nucleic acid constructs mean constructs derived from an alien species, or, if derived from the same species, a construct substantially modified from its native form in composition and / or genomic locus by intentional human intervention. Heterologous nucleic acid constructs include, but are not limited to, the recombinant nucleotide constructs introduced into plants or their plant parts, for example, by transformation methods or subsequently by transgenic plants and another target plant breeding. In one aspect, the promoter used is heterologous to the sequence driven by the promoter. On the other hand, the promoter used is heterologous to tobacco. On the other hand, the promoter used is natural to tobacco.

[0152] In one aspect, the modified tobacco plant is a cisgenic plant. As used herein, "cis-occurrence" or "cisgenic" refers to the genetic modification of plant, plant cell or plant genome, wherein all components (for example, promoter, donor nucleic acid, selection gene) only have plant origin (that is, do not use the assembly of non-plant origin). In one aspect, the modified plant, plant cell or plant genome provided are cisgenic. The cisgenic plant, plant cell and plant genome provided can produce ready-to-use tobacco strains. On the other hand, the modified tobacco plant provided does not comprise non-tobacco genetic material or sequence.

[0153] As used herein, "gene expression" refers to the biosynthesis or production of a gene product, including the transcription and / or translation of the gene product.

[0154] Also provided herein are compositions and methods for overexpressing one or more polypeptides of the Nic1b_ERF locus in plants, particularly plants of the genus Nicotiana, including various commercial varieties of tobacco plants.

[0155] In one aspect, the disclosure provides a recombinant DNA construct comprising a promoter, the promoter being functional in tobacco cells and operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207 and 222-228, and fragments thereof. Also provided are transgenic tobacco plants or parts thereof, cured tobacco materials or tobacco products comprising these recombinant DNA constructs. In one aspect, these transgenic plants, cured tobacco materials or tobacco products comprise increased levels of nicotine compared to control tobacco plants not containing the recombinant DNA construct. Also provided is a method for increasing the nicotine level of a tobacco plant, the method comprising transforming a tobacco plant with any of these recombinant DNA constructs.

[0156] In one aspect, for each transgenic or mutant line carrying a Nic1b-ERF-associated transgene or mutation, a combination of such transgenic or mutant lines and a mutation or transgenic event for one or more ERF genes of the Nic2 locus is also provided. Such one or more Nic2 ERF genes include, but are not limited to, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (Shoji et al., Plant Cell, (10): 3390-409 (2010)). Of particular interest and provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, five or more, six or more, or seven or more Nic1b ERF-like genes (e.g., NCG1, NCG11, NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168). Of further particular interest and provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) and transgenic or mutagenic suppression of ERF189, ERF115, or both. Also provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) and transgenic or mutagenic suppression of ERF189. In another aspect, provided herein are plants having transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (e.g., NCG12, NCG15, NCG16, NCG17, ERF101, ERF110, ERF16, ERF130) in combination with transgenic or mutagenic suppression of ERF115.Of particular interest and provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or four or more Nic1b ERF-like genes (NCG1, NCG12, NCG15, NCG16, NCG17) and one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168). Further of particular interest and provided herein are plants having a combination of transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) and transgenic or mutagenic suppression of ERF189, ERF115, or both. Also provided herein are plants having a transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) in combination with a transgenic or mutagenic suppression of ERF189. In another aspect, provided herein are plants having a transgenic or mutagenic suppression of one or more, two or more, three or more, or all four Nic1b ERF-like genes (NCG12, NCG15, NCG16, NCG17) in combination with a transgenic or mutagenic suppression of ERF115.

[0157] In another aspect, a tobacco plant having a transgenic or mutagenic suppression of NCG12 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, a tobacco plant having a transgenic or mutagenic suppression of NCG15 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, a tobacco plant having a transgenic or mutagenic suppression of NCG16 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, a tobacco plant having a transgenic or mutagenic suppression of NCG17 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, a tobacco plant having a transgenic or mutagenic suppression of ERF101 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, a tobacco plant having a transgenic or mutagenic suppression of ERF110 and a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168) is provided. In another aspect, tobacco plants are provided having transgenic or mutagenic suppression of ERF16 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168).In another aspect, tobacco plants are provided having transgenic or mutagenic suppression of ERF130 and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic2 ERF genes (ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168).

[0158] In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of one or more Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking the transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of one or more Nic1b_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0159] In one aspect, a tobacco plant having transgenic or mutagenic inhibition of two or more Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having transgenic or mutagenic inhibition of two or more Nic1b_ERF genes and transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0160] In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of three or more Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking the transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of three or more Nic1b_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0161] In one aspect, a tobacco plant having transgenic or mutagenic inhibition of four or more Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having transgenic or mutagenic inhibition of four or more Nic1b_ERF genes and transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0162] In one aspect, a tobacco plant having transgenic or mutagenic inhibition of five or more Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having transgenic or mutagenic inhibition of five or more Nic1b_ERF genes and transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0163] In one aspect, a tobacco plant having transgenic or mutagenic inhibition of six or more Nic1b_ERF genes is provided. In one aspect, the plant comprises lower nicotine or total alkaloid levels compared to a control plant lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having transgenic or mutagenic inhibition of six or more Nic1b_ERF genes and transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0164] In one aspect, a tobacco plant having less than two transgenic or mutagenic inhibitions of Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having less than two transgenic or mutagenic inhibitions of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic inhibitions of Nic2_ERF genes is provided.

[0165] In one aspect, a tobacco plant having less than three transgenic or mutagenic inhibitions of Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having less than three transgenic or mutagenic inhibitions of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic inhibitions of Nic2_ERF genes is provided.

[0166] In one aspect, a tobacco plant having less than four transgenic or mutagenic inhibition of Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having less than four transgenic or mutagenic inhibition of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic inhibition of Nic2_ERF genes is provided.

[0167] In one aspect, a tobacco plant having less than five transgenic or mutagenic inhibitions of Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having less than five transgenic or mutagenic inhibitions of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic inhibitions of Nic2_ERF genes is provided.

[0168] In one aspect, a tobacco plant having less than six transgenic or mutagenic inhibitions of Nic1b_ERF genes is provided. In one aspect, such plants contain lower nicotine or total alkaloid levels compared to control plants lacking transgenic or mutagenic inhibition under comparable conditions. In one aspect, a tobacco plant having less than six transgenic or mutagenic inhibitions of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic inhibitions of Nic2_ERF genes is provided.

[0169] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of one or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels compared to control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of one or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0170] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of two or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels compared to control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of two or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0171] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of three or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of three or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0172] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of four or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of four or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0173] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of five or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels compared to control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of five or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0174] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of six or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of six or more Nic1b_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic2_ERF genes is provided.

[0175] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than two Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than two Nic1b_ERF genes and a transgenic or mutagenic overexpression of less than two, less than three, less than four, less than five, less than six, or less than seven Nic2_ERF genes is provided.

[0176] In one aspect, a tobacco plant having less than three transgenic or mutagenic overexpression of Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having less than three transgenic or mutagenic overexpression of Nic1b_ERF genes, and less than two, less than three, less than four, less than five, less than six, or less than seven transgenic or mutagenic overexpression of Nic2_ERF genes is provided.

[0177] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than four Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than four Nic1b_ERF genes and a transgenic or mutagenic overexpression of less than two, less than three, less than four, less than five, less than six, or less than seven Nic2_ERF genes is provided.

[0178] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than five Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than five Nic1b_ERF genes and a transgenic or mutagenic overexpression of less than two, less than three, less than four, less than five, less than six, or less than seven Nic2_ERF genes is provided.

[0179] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than six Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels than control plants lacking the transgenic or mutagenic overexpression under comparable conditions. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of less than six Nic1b_ERF genes and a transgenic or mutagenic overexpression of less than two, less than three, less than four, less than five, less than six, or less than seven Nic2_ERF genes is provided.

[0180] In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of one or more Nic2_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of two or more Nic2_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of three or more Nic2_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic inhibition of four or more Nic2_ERF genes and a transgenic or mutagenic inhibition of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, tobacco plants having transgenic or mutagenic suppression of five or more Nic2_ERF genes and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes are provided. In one aspect, any of the foregoing plants comprises lower nicotine or total alkaloid levels compared to control plants not having such Nic2_ERF or Nic1b_ERF transgenic or mutagenic suppression under comparable conditions.

[0181] In one aspect, tobacco plants having less than two Nic2_ERF genes transgenic or mutagenic inhibition, and less than two, less than three, less than four, less than five, or less than six Nic1b_ERF genes transgenic or mutagenic inhibition are provided. In one aspect, tobacco plants having two or more Nic2_ERF genes transgenic or mutagenic inhibition, and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes transgenic or mutagenic inhibition are provided. In one aspect, tobacco plants having three or more Nic2_ERF genes transgenic or mutagenic inhibition, and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes transgenic or mutagenic inhibition are provided. In one aspect, tobacco plants having four or more Nic2_ERF genes transgenic or mutagenic inhibition, and one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes transgenic or mutagenic inhibition are provided. In one aspect, tobacco plants having transgenic or mutagenic suppression of five or more Nic2_ERF genes and transgenic or mutagenic suppression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes are provided. In one aspect, any of the foregoing plants comprises lower nicotine or total alkaloid levels compared to control plants not having Nic2_ERF or Nic1b_ERF transgenic or mutagenic suppression under comparable conditions.

[0182] In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of one or more Nic2_ERF genes, and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of two or more Nic2_ERF genes, and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of three or more Nic2_ERF genes, and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having a transgenic or mutagenic overexpression of four or more Nic2_ERF genes, and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, a tobacco plant having transgenic or mutagenic overexpression of five or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels compared to control plants not having such Nic2_ERF or Nic1b_ERF transgenic or mutagenic overexpression under comparable conditions.

[0183] In one aspect, there is provided a tobacco plant having a transgenic or mutagenic overexpression of less than two Nic2_ERF genes and a transgenic or mutagenic overexpression of less than two, less than three, less than four, less than five or less than six Nic1b_ERF genes. In one aspect, there is provided a tobacco plant having a transgenic or mutagenic overexpression of two or more Nic2_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one aspect, there is provided a tobacco plant having a transgenic or mutagenic overexpression of three or more Nic2_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one aspect, there is provided a tobacco plant having a transgenic or mutagenic overexpression of four or more Nic2_ERF genes and a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes. In one aspect, a tobacco plant having transgenic or mutagenic overexpression of five or more Nic2_ERF genes and transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more, or five or more Nic1b_ERF genes is provided. In one aspect, such plants contain higher nicotine or total alkaloid levels compared to control plants not having Nic2_ERF or Nic1b_ERF transgenic or mutagenic overexpression under comparable conditions.

[0184] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: 222 and 226, and the tobacco plant further having a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0185] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID No 73, 83, 84 and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0186] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of one or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of one or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0187] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of one or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: 222 and 226, and said tobacco plant also having a transgenic or mutagenic suppression of one or more Nic1b_ERF genes, said genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID No 73, 83, 84 and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0188] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0189] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of two or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0190] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0191] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes, the genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of three or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0192] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than two Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has transgenic or mutagenic suppression of less than two Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206, and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0193] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than two Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has transgenic or mutagenic suppression of less than two Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0194] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than three Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of less than three Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0195] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than three Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of less than three Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0196] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than four Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of less than four Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0197] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than four Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has transgenic or mutagenic suppression of less than four Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0198] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than five Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159 and 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has a transgenic or mutagenic suppression of less than five Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0199] In one aspect, a tobacco plant is provided having a transgenic or mutagenic suppression of one or two Nic2_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and said tobacco plant also having a transgenic or mutagenic suppression of less than five Nic1b_ERF genes, said genes comprising a sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53 and 54. In one aspect, a tobacco plant is provided having transgenic or mutagenic suppression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of 222 and 226, and the tobacco plant also has transgenic or mutagenic suppression of less than five Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID No 73, 83, 84, and 87-89. In one aspect, the plant described in this paragraph comprises lower nicotine or total alkaloid levels compared to a control plant lacking such transgenic or mutagenic suppression under comparable conditions.

[0200] In one aspect, the mutagenesis inhibition of the Nic2_ERF gene does not contain a deletion of the entire Nic2_ERF gene or the entire Nic2_ERF coding region. In one aspect, the mutagenesis inhibition of the Nic2_ERF gene contains a deletion of the entire Nic2_ERF gene or the entire Nic2_ERF coding region. In one aspect, the mutagenesis inhibition of the Nic1b_ERF gene does not contain a deletion of the entire Nic1b_ERF gene or the entire Nic1b_ERF coding region. In one aspect, the mutagenesis inhibition of the Nic1b_ERF gene contains a deletion of the entire Nic1b_ERF gene or the entire Nic1b_ERF coding region.

[0201] In one aspect, a tobacco plant is provided having a transgenic or mutagenized overexpression of one or two Nic2_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenized overexpression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes, the genes comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, 202-205. In one aspect, a tobacco plant is provided having a transgenic or mutagenic overexpression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: 222 and 226, and the tobacco plant further having a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID No 73, 83, 84, 87-89, 180, 181, 206 and 207. In one aspect, the plant described in this paragraph comprises a higher level of nicotine or total alkaloids than a control plant lacking such transgenic or mutagenic overexpression under comparable conditions.

[0202] In one aspect, a tobacco plant is provided, which has a transgenic or mutagenic overexpression of one or two Nic2_ERF genes, wherein the genes comprise a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: 208, 212, 215 and 219, and the tobacco plant also has a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes, wherein the genes comprise a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of: SEQ ID No 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54. In one aspect, a tobacco plant is provided having a transgenic or mutagenic overexpression of one or two Nic2_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: 222 and 226, and the tobacco plant also has a transgenic or mutagenic overexpression of one or more, two or more, three or more, four or more or five or more Nic1b_ERF genes encoding a polypeptide comprising a sequence at least 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID No 73, 83, 84 and 87-89. In one aspect, the plant described in this paragraph comprises a higher level of nicotine or total alkaloids than a control plant lacking such transgenic or mutagenic overexpression under comparable conditions.

[0203] In one aspect, lower nicotine or total alkaloid levels are at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% lower than control plants under comparable conditions.

[0204] In one aspect, higher nicotine or total alkaloid levels refer to an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700% or 800% compared to control plants under comparable conditions.

[0205] In one aspect, the recombinant DNA construct or expression cassette may also include a selectable marker gene for selecting transgenic cells. Selectable marker genes include but are not limited to genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds, such as glufosinate, bromoxynil, imidazolinone and 2,4-dichlorophenoxyacetic acid (2,4-D). Other selectable markers include phenotypic markers (such as beta-galactosidase) and fluorescent proteins (such as green fluorescent protein (GFP)).

[0206] In one aspect, the recombinant DNA construct or expression cassette comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-preferred promoter (eg, a leaf-specific or root-specific promoter). Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter disclosed in U.S. Pat. No. 6,072,050 and other constitutive promoters; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), etc. Exemplary chemically inducible promoters include the tobacco PR-1a promoter activated by salicylic acid. Other target chemically inducible promoters include steroid responsive promoters (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14 (2): 247-257 glucocorticoid inducible promoters) and tetracycline inducible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156).Other exemplary promoters that can be used are promoters responsible for heat-regulated gene expression, light-regulated gene expression (e.g., pea rbcS-3A; maize rbcS promoter; the chlorophyll alb binding protein gene found in pea; or the Arabssu promoter), hormone-regulated gene expression (e.g., the abscisic acid (ABA) response sequence of the Em gene of wheat; the ABA-inducible HVA1 and HVA22 and rd29A promoters of barley and Arabidopsis; and wound-induced gene expression ( For example, gene expression of wunl), organ-specific gene expression (e.g., gene expression of a tuber-specific storage protein gene); the 23-kDa zein gene from maize; or lentil (β-phaseolin gene), or a pathogen-inducible promoter (e.g., the PR-1, prp-1, or β-1,3 glucanase promoter, the fungus-inducible wirla promoter from wheat, and the nematode-inducible promoters TobRB7-54 and Hmg-1 from tobacco and parsley, respectively).

[0207] In one aspect, the tobacco plant provided also comprises the expression of the increase or reduction of the activity of the gene participating in nicotine biosynthesis or transport.The gene related to nicotine biosynthesis includes but is not limited to arginine decarboxylase (ADC), methyl putrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosyl anthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolinate phosphoribosyltransferase (QPT) and S-adenosylmethionine synthetase (SAMS).Although two kinds of candidate genes (A622 and NBB1) have been proposed, the nicotine synthase of the condensation step between catalysis nicotinic acid derivative and methylpyrroline cation has not yet been explained.Referring to US2007 / 0240728A1 and US2008 / 0120737A1.A622 encodes isoflavone reductase sample protein.In addition, several transporters can participate in the transport of nicotine. A transporter gene called MATE has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

[0208] In one aspect, the tobacco plant provided also comprises an increase or decrease in the level of mRNA, protein or both of one or more genes compared to a control tobacco plant, the gene encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB1. On the other hand, the tobacco plant provided also comprises a transgenic that directly inhibits the expression of one or more genes, the gene encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB1. On the other hand, the tobacco plant provided also comprises a transgenic or mutation that inhibits the expression or activity of one or more genes, the gene encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622 and NBB1. In another aspect, a tobacco plant is provided that further comprises a transgene that overexpresses one or more genes encoding a product selected from the group consisting of PMT, MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

[0209] Also disclosed is the use of any suitable transformation method known in the art to transform tobacco plants with the recombinant construct or expression cassette. The method of introducing a polynucleotide sequence into tobacco plants is known in the art, including but not limited to stable transformation methods, transient transformation methods and virus-mediated methods."stable transformation" refers to the transformation of the target nucleotide construct introduced into a plant that is integrated into the plant genome and can be inherited by its offspring."transient transformation" refers to the introduction of a sequence into a plant and only transiently expressed or only transiently present in the plant.

[0210] Suitable methods for introducing polynucleotides into plant cells of the present disclosure include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol. 153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al. (1996) EMBO J. 3:2717-2722). al. (1995) in Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6: 923-926).See also Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, NY), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation).

[0211] On the other hand, the recombinant construct or expression cassette can be introduced into the plant by contacting the plant with a virus or viral nucleic acid. Typically, such methods involve incorporating the expression cassette of the present disclosure into a viral DNA or RNA molecule. It is recognized that promoters for expression cassettes also encompass promoters for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing proteins encoded therein that are related to viral DNA or RNA molecules are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta et al. (1996) Molecular Biotechnology 5: 209-221.

[0212] Any plant tissue that can be subsequently propagated using cloning methods, whether by organogenesis or embryogenesis, can be transformed with recombinant constructs or expression cassettes." Organogenesis" means the process of developing buds and roots from the center of meristems in sequence. "Embryogenesis" means the process of developing buds and roots together in a consistent manner (rather than sequentially) from somatic cells or gametes. Exemplary tissues suitable for the various transformation schemes include, but are not limited to, callus, existing meristems (e.g., apical meristems, axillary buds, and root meristems) and induced meristems (e.g., cotyledon meristems and hypocotyl meristems), hypocotyls, cotyledons, leaf disks, pollen, embryos, etc.

[0213] In one aspect, the tobacco plant provided is from the tobacco type selected from the group consisting of: flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark flue-cured tobacco, Galpao tobacco and oriental tobacco. On the other hand, the tobacco plant provided is from the tobacco type selected from the group consisting of: burley tobacco, Maryland tobacco and dark tobacco.

[0214] In one aspect, the tobacco plant provided is in a cured tobacco background or exhibits one or more cured tobacco characteristics as described herein. Cured tobacco (also referred to as Virginia or bright tobacco) accounts for about 40% of the world's tobacco production. Because cured tobacco reaches a golden to dark orange color during the curing process, cured tobacco is also commonly referred to as "bright tobacco". Cured tobacco has a light, bright aroma and taste. Cured tobacco usually has a high sugar content and a low oil content. The main cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the United States. In one aspect, low alkaloid or low nicotine tobacco plants or seeds are provided in a flue-cured tobacco background selected from the group consisting of CC 13, CC 27, CC 33, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K 326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC606, NC 71, NC 72, NC 92. PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety substantially derived from any of the foregoing varieties.In another aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a flue-cured tobacco background selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair 944, Ox 207, Ox414NF, Reams 126, Reams 713, Reams 744, RG 8, RG In another aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety or strain is in any flue-cured tobacco background selected from the group consisting of K326, K346 and NC196. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a flue-cured tobacco variety selected from the varieties listed in Table 15 and any variety substantially derived from any of the foregoing varieties. See WO 2004 / 041006 A1.

[0215] In one aspect, the tobacco plants provided are in an air-cured tobacco background or exhibit one or more air-cured tobacco characteristics described herein. Air-cured tobacco includes burley, Maryland and dark tobacco. The commonality is that the curing is mainly without artificial heat and humidity sources. Burley is light to dark brown in color, high in oil content and low in sugar content. Burley is air-cured in barns. The main countries where burley is grown are Argentina, Brazil, Italy, Malawi, and the American Maryland tobacco is very fluffy, has good burning characteristics, low nicotine and neutral flavor. The main Maryland growing countries include the United States and Italy. In one aspect, a low alkaloid or low nicotine tobacco plant or seed is provided in a burley background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC2000, Tn 86, Tn 90, Tn 97, R 610, R 630, R 711, R 712, NCBH 129, Bu 21×Ky 10, HB04P, Ky14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509. In one aspect, tobacco plant or seed or modified tobacco plant or seed provided herein is selected from the tobacco variety listed in the table 16 and the burley tobacco variety of any kind derived from the aforementioned variety basically.On the other hand, low alkaloid or low nicotine tobacco plant, seed, hybrid, variety or strain are in any burley tobacco background selected from the group consisting of: TN 90, KT 209, KT 206, KT 212 and HB4488.On the other hand, low alkaloid or low nicotine tobacco plant or seed provided are in the Maryland tobacco background selected from the group consisting of: Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341.On the other hand, tobacco plant or seed or modified tobacco plant or seed provided herein is selected from the tobacco variety listed in the table 17 and the Maryland tobacco variety of any kind derived from the aforementioned variety basically.

[0216] In one aspect, the tobacco plant provided is in a dark air-cured tobacco background or exhibits one or more dark air-cured tobacco grass characteristics as described herein. The difference between air-cured black tobacco and other types is mainly that the dark air-cured tobacco has a medium to dark brown color and a unique fragrance through its curing process. Dark air-cured tobacco is mainly used to produce chewing tobacco and snuff. In one aspect, the low alkaloid or low nicotine tobacco plant or seed provided is in a dark air-cured tobacco background selected from the group consisting of Sumatra, Jatim, Dominican, Cubano, Besuki, Onesucker, Green River, Virginia sun-cured and Paraguan Passado, and any variety derived substantially from any of the aforementioned varieties.

[0217] In one aspect, the tobacco plant provided is in a dark-cured cured tobacco background or exhibits one or more dark-cured cured tobacco characteristics as described herein. Dark-cured cured tobacco is usually cured on the floor of a closed curing barn with a low-burning wood fire. Their leaves have a low sugar content but a high nicotine content. Dark-cured cured tobacco is used to make tubular blends, cigarettes, chewing tobacco, snuff, and strong tobacco-flavored cigars. The main growing areas of dark-cured cured tobacco are Tennessee, Kentucky, and Virginia, USA. In one aspect, the low alkaloid or low nicotine tobacco plant or seed provided is in a dark-cured cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a dark-cured cured tobacco variety selected from the tobacco varieties listed in Table 18 and any variety substantially derived from any of the aforementioned varieties.

[0218] In one aspect, the tobacco plant provided is in oriental tobacco background or shows one or more oriental tobacco features as herein described.Because oriental tobacco is typically grown in the eastern Mediterranean region, for example Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy and Romania, they are also referred to as Greek tobacco, aromatherapy tobacco and Turkish tobacco.Now the distinctive little plant of oriental variety and leaf size and its unique fragrance feature are the result of plant development poor adaptation soil and stress climate conditions in the past centuries.In one aspect, low alkaloid or low nicotine tobacco plant or seed provided herein are in oriental tobacco background selected from lower group: Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir and basically derived from any kind in aforementioned variety. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is an Oriental variety selected from the tobacco varieties listed in Table 19 and any variety substantially derived from any of the foregoing varieties.

[0219] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a cigar variety selected from the tobacco varieties listed in Table 20 and any variety substantially derived from any of the foregoing varieties.

[0220] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is a tobacco variety selected from the tobacco varieties listed in Table 21 and any variety substantially derived from any of the foregoing varieties.

[0221] In one aspect, the modified tobacco plant, seed or cell described herein is from a variety selected from the group consisting of tobacco varieties listed in Table 15, Table 16, Table 17, Table 18, Table 19, Table 20 and Table 21.

[0222] In one aspect, the tobacco plants, seeds, hybrids, varieties or lines described herein (which may be low alkaloid, low nicotine, high alkaloid or high nicotine) are substantially derived from or are under the genetic background of tobacco: BU64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K730, KD 730, KD 731, KD 732, KD 733, KD 734, KD 735, KD 736, KD 737, KD 738, KD 739, KD 739, KD 730, KD 731, KD 732, KD 733, KD 734, KD 735, KD 736, KD 737, KD 738 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14 x L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow LeafMadole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, `Perique`tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG17, RG 81, RG H51, RGH 4, RGH 51, RS1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950、TR(Tom Rosson)Madole、VA 309、orVA359、Maryland609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art.

[0223] All specific varieties of the dark air-aged, white burley, Maryland, dark roasted, or oriental types mentioned above are listed for exemplary purposes only. Any other dark air-aged, white burley, Maryland, dark roasted, or oriental varieties are also contemplated in this application.

[0224] The colony of described tobacco plant is also provided.In one aspect, the planting density of the colony of tobacco plant is about 5,000 to about 8000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8000, about 7200 to about 8000, or about 7600 to about 8000 strains of plants.On the other hand, the colony of tobacco plant is in the soil type low to medium fertility.

[0225] The container of the seed of described tobacco plant is also provided.The container of tobacco seed of the present disclosure can comprise the seed of any quantity, weight or volume.For example, container can hold at least or be greater than about 100,200,300,400,500,600,700,800,900,1000,1500,2000,2500,3000,3500,4000 or more seed.Perhaps, described container can hold at least or be greater than about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seed.The container of tobacco seed can be any container obtainable in this area.As non-limiting embodiments, container can be box, bag, bag, pouch, film roll (taperoll), pipe or bottle.

[0226] Also provided is a cured tobacco material made from the low alkaloid or low nicotine tobacco plant. Also provided is a cured tobacco material with a higher level of total alkaloids or nicotine made from the tobacco plant.

[0227] "Curing" is an aging process that reduces moisture and destroys chlorophyll, making tobacco leaves golden yellow, and starch is converted into sugar by this process. Therefore, compared with the green leaves harvested, cured tobacco has a higher reducing sugar content and a lower starch content. In one aspect, the green leaf tobacco provided can be cured using conventional means, for example, baking and curing, barn curing, fire-baked curing, air-drying and curing or sun-curing. See, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for descriptions of different types of curing methods. Cured tobacco is usually aged for several years (for example, two to five years) in a wooden barrel (for example, a large barrel (Hogshead)) or a cardboard box with a moisture content ranging from 10% to about 25% under pressure conditions. See U.S. Patent Nos. 4,516,590 and 5,372,149. Then, the cured and aged tobacco can be further processed. Further processing includes conditioning tobacco under various temperatures, pasteurization and fermentation under vacuum with or without the introduction of steam. Typically, fermentation is characterized by high initial moisture content, heat release and 10-20% dry weight loss. For example, see U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, aged and fermented tobacco (e.g., cutting, shredding, puffing or mixing) can be further processed. For example, see U.S. Patent Nos. 4,528,993; 4,660,577; and 4,987,907. In one aspect, the cured tobacco material of the present disclosure is sun-cured. In another aspect, the cured tobacco material of the present disclosure is flue-cured, air-cured, or fire-cured.

[0228] Tobacco material obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to prepare tobacco products.As used herein, "tobacco product" is defined as any product made or derived from tobacco intended for human use or consumption.

[0229] Tobacco products provided include, but are not limited to, cigarette products (e.g., cigarettes and bidis), cigar products (e.g., cigar wrappers and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, sheets, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US2006 / 0191548.

[0230] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". A cigarette "rod" includes cigarette paper, a filter, a filter rod (for containing filter material), a tipping paper that holds the cigarette paper (including filler) to the filter, and all glue that holds these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other tobacco flavors that can be rolled into cigarette paper along with tobacco), (3) casing, (4) flavoring agents, and (5) all other additives (mixed into tobacco and substitutes and rolled into cigarettes).

[0231] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco waste, which is processed into sheet form and cut into rods to simulate tobacco. In addition to cost savings, reconstituted tobacco is very important for its contribution to the flavor of cigarettes by processing flavors using a reaction between ammonia and sugars.

[0232] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed by expansion with a suitable gas so that the tobacco is "expanded," resulting in reduced density and greater filling capacity. It reduces the weight of the tobacco used in cigarettes.

[0233] Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking products, particularly those including filter elements, wherein the rod of the smokeable material includes cured tobacco in the tobacco blend. In one aspect, the tobacco products of the present disclosure are selected from the group consisting of cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigars, cigarettes, chewing tobacco, tobacco leaves, hookahs, cigarette shreds and shredded tobacco. On the other hand, the tobacco products of the present disclosure are smokeless tobacco products. Smokeless tobacco products do not burn, including but not limited to chewing tobacco, moist smokeless tobacco, snus and dry snuff. Chewing tobacco is a coarsely divided tobacco leaf, which is typically packaged in a large bag-shaped package and used in a plug or twisted manner. Moist smokeless tobacco is moist, finer tobacco, which is provided in loose form or in a pouch form, and is typically packaged in a round can, and is used as a pinch or pouch placed between the cheeks and gums of an adult tobacco consumer. Snus is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco placed in the mouth or nose. In another aspect, the tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of electronically heated cigarettes, electronic cigarettes, and electronic vaporization devices.

[0234] In one aspect, the tobacco product of the present disclosure can be a blended tobacco product. In another aspect, the tobacco product of the present disclosure can be a low-nicotine tobacco product. In another aspect, the tobacco product of the present disclosure can contain nornicotine at a level of less than about 3 mg / g. For example, the content of nornicotine in such preparations can be 3.0 mg / g, 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, 750 μg / g, 500 pg / g, 250 pg / g, 100 pg / g, 75 pg / g, 50 pg / g, 25 pg / g, 10 pg / g, 7.0 pg / g, 5.0 pg / g, 4.0 pg / g, 2.0 pg / g, 1.0 pg / g, 0.5 pg / g, 0.4 pg / g, 0.2 pg / g, 0.1 pg / g, 0.05 pg / g, 0.01 pg / g, or undetectable.

[0235] In one aspect, provided cured tobacco materials or tobacco products comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.9%, 6.1%, 6.2%, 6 ... 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8% and 9%. In another aspect, a cured tobacco material or tobacco product is provided comprising an average nicotine or total alkaloid level selected from the group consisting of about 0.01%-0.02%, 0.02%-0.05%, 0.05%-0.75%, 0.75%-0.1%, 0.1%-0.15%, 0.15%-0.2%, 0.2%-0.3%, 0.3%-0.35%, 0.35%-0.4%, 0.4%-0.5%, 0.5%-0.6%, 0.6%-0.7%, 0.7%-0.8%, 0.8%-0.9%, 0.9%-1%, 1%-1.1%, 1.1%-1.2%, 1.2%-1.3%, 1.4%-1.5%, 1.6%-1.7%, 1.8%-1.9%, 1.9%-2.0%, 1.1%-2.1%, 1.2%-2.3%, 1.3%-2.4%, 1.5%-2.6%, 1.6%-2.7%, 1.8%-2.9%, 1.9%-2.1%, 1.8%-2. 1.2%-1.3%, 1.3%-1.4%, 1.4%-1.5%, 1.5%-1.6%, 1.6%-1.7%, 1.7%-1.8%, 1.9%-2%, 2%-2.1%, 2.1%-2.2%, 2.2%-2.3%, 2.3%-2.4%, 2.4%-2.5%, 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, 2.9%-3%, 3%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, 3.4%-3.5% and 3.5%-3.6%. In another aspect, provided cured tobacco materials or tobacco products comprise an average nicotine or total alkaloid level selected from the group consisting of about 0.01%-0.1%, 0.02%-0.2%, 0.03%-0.3%, 0.04%-0.4%, 0.05%-0.5%, 0.75%-1%, 0.1%-1.5%, 0.15%-2%, 0.2%-3% and 0.3%-3.5% on a dry weight basis.

[0236] The disclosure also provides the method for the tobacco strain, cultivar or variety that comprises the total alkaloid of required level or nicotine (for example low nicotine or nicotine-free) for breeding.Can be bred by any known method.DNA fingerprint, SNP mapping (mapping), haplotype mapping or similar techniques can be used for marker assisted selection (MAS) breeding program with desired proterties or allele transfer or breeding in tobacco plant.For example, breeder can use F1 hybrid plant to produce isolated population in F2 generation or backcross generation, or further hybridize F1 hybrid plant with other donor plants with agronomically required genotype.Can use one of the technology known in the art or listed herein to screen the required agronomy proterties or required chemical characteristics of F2 generation or backcross generation plant.According to expected genetic pattern or used MAS technology, can carry out self-pollination of selected plant before each backcross cycle to help identify required individual plant.Can repeat backcrossing or other breeding programs until the required phenotype of the recurrent parent is restored. The recurrent parent in the present disclosure can be a flue-cured variety, a burley variety, a dark air-cured variety, a dark flue-cured variety or an oriental tobacco variety. Other breeding techniques are found in, for example, Wernsman, EA and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY, which is incorporated herein by reference in its entirety.

[0237] The result of the plant breeding program using the described tobacco plant comprises useful lines, cultivars, kinds, offspring, inbred lines and hybrids of the present disclosure.As used herein, the term "kind" refers to a plant population with constant characteristics, which distinguishes it from other plants of the same species.Though not always, kind can be sold as a commodity usually.Although having one or more distinctive traits, the characteristic of kind is that the overall variation between the individual in this kind is also very small." pure line " kind can be produced from asexual reproduction of a single parent by self-pollination and selection for several generations, or using tissue or cell culture techniques.Kind can be derived from another line or kind basically. According to the International Convention for the Protection of New Varieties of Plants (December 2, 1961; November 10, 1972, October 23, 1978 and March 19, 1991, revised in Geneva), variety is "basically derived from" the initial variety, if: a) it is mainly derived from the initial variety, or derived from a variety mainly derived from the initial variety, while retaining the expression of the basic characteristics produced by the genotype or genotype combination of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the difference produced by the derivation, it is consistent with the initial variety in the expression of the basic characteristics produced by the genotype or genotype combination of the initial variety. For example, by selecting natural or induced mutants, somatic cell clone variants, variant individuals from the plant of the initial variety, backcrossing or conversion, a basically derived variety can be obtained. The second tobacco variety from which the first tobacco variety and the first variety are basically derived is considered to have basically the same genetic background. "Strain" different from variety most usually represents a group of plants used for non-commercial purposes, for example in plant research. Lines typically show little overall variation among individuals for one or more traits of interest, although there may be some variation among individuals for other traits.

[0238] In one aspect, the disclosure provides a method for introgressing low nicotine traits into tobacco varieties, the method comprising: (a) hybridizing the first tobacco variety comprising low nicotine traits with the second tobacco variety not having low nicotine traits to produce one or more offspring tobacco plants; (b) genotyping the one or more offspring tobacco plants for a polymorphic marker linked to the low nicotine trait, wherein the polymorphic marker is in a chromosome interval, and the chromosome interval flanking any two of the SNP markers selected from the group below: SEQ ID No.125-145 and 193-201; and (c) selecting the offspring tobacco plants comprising low nicotine traits. On the other hand, these methods also include backcrossing the offspring tobacco plants and the second tobacco variety selected. In yet another aspect, these methods also include: (d) hybridizing the offspring plants selected with themselves or with the second tobacco variety to produce one or more further offspring tobacco plants; and (e) selecting another offspring tobacco plant comprising low nicotine traits. In one aspect, the selection of step (e) includes marker-assisted selection. In one aspect, these methods produce a single gene conversion comprising low nicotine traits. In one aspect, these methods produce a single gene transformation comprising a Nic1b_ERF gene introgression. In one aspect, the second tobacco variety is an elite variety. In another aspect, the genotyping step of these methods relates to one or more molecular marker assays. In another aspect, the polymorphic marker used in the method comprises a polymorphism selected from the group consisting of a single nucleotide polymorphism (SNP), an insertion or deletion (Indel) in a DNA sequence, a simple sequence repeat (SSR) of a DNA sequence, a restriction fragment length polymorphism (RFLP) and a tag SNP. In another aspect, the offspring tobacco plant selected comprises a shorter chromosome introgression at the Nic1b_ERF locus compared to LA Burley 21.

[0239] In another aspect, the present disclosure provides a method for introgressing a low nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety comprising a low nicotine trait with a second tobacco variety that does not have the low nicotine trait to produce one or more offspring tobacco plants; (b) genotyping the one or more offspring tobacco plants for a polymorphic marker linked to the low nicotine trait, wherein the polymorphic marker is within 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less than 0.5 cM of any one of the SNP markers selected from the group consisting of SEQ ID Nos. 125-145 and 193-201 or within any locus having a sequence selected from the group consisting of SEQ ID Nos. 1, 3-37, 146, 149, 152-156, 202, 203 and 184-186; and (c) selecting offspring tobacco plants comprising the low nicotine trait. In one aspect, the method comprises simultaneously or in parallel selecting one or more molecular markers associated with or closely linked to the Nic1b_ERF locus and one or more molecular markers associated with or closely linked to the Nic2 locus.

[0240] In one aspect, the disclosure provides a method for selecting a tobacco plant with a low nicotine trait, the method comprising: (a) isolating nucleic acid from a tobacco germplasm collection; (b) determining one or more markers in the nucleic acid that are closely linked to the Nic1b_ERF locus; and (c) selecting a tobacco plant with a low nicotine trait based on the marker determination. In one aspect, the one or more markers determined to be closely linked to the Nic1b_ERF locus are within about 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less than 0.5 cM of any one of the SNP markers selected from the group consisting of SEQ ID Nos. 125-145 and 193-201, or within any locus having a sequence selected from the group consisting of SEQ ID Nos. 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54, 153, 154, 158, 159, and 202-205. On the other hand, the method also includes measuring one or more marks closely linked to the Nic2 locus. In one aspect, the one or more marks closely linked to the Nic2 locus measured are about 20cM, 10cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.5cM or less than 0.5cM of any polymorphic locus in one of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168. In one aspect, the method also includes measuring the nicotine level of the plant selected to determine the low nicotine trait.

[0241] Also disclosed is a method for introgressing low nicotine traits into tobacco varieties, the method comprising: (a) hybridizing a first tobacco variety comprising low nicotine traits with a second tobacco variety not having low nicotine traits to produce one or more offspring tobacco plants; (b) genotyping the one or more offspring tobacco plants for polymorphic markers linked to the low nicotine trait, wherein the polymorphic markers are in a chromosome interval, and the chromosome interval flanking any two of the SNP markers selected from the group consisting of: SEQ ID No.125-145 and 193-201; and (c) selecting offspring tobacco plants comprising low nicotine traits. In one aspect, these methods produce a single gene conversion comprising low nicotine traits. On the one hand, these methods produce a single gene conversion comprising Nic2 gene introgression. In one aspect, the second tobacco variety is an elite variety. On the other hand, the genotyping step of these methods relates to one or more molecular marker assays. On the other hand, the polymorphic markers used in the method include a polymorphism selected from the group consisting of a single nucleotide polymorphism (SNP), an insertion or deletion (Indel) in a DNA sequence, a simple sequence repeat (SSR) of a DNA sequence, a restriction fragment length polymorphism (RFLP) and a tag SNP. On the other hand, the offspring tobacco plant selected comprises a shorter chromosome deletion at the Nic2 locus compared to LA Burley 21.

[0242] As used herein, a "locus" is a chromosomal region where a polymorphic nucleic acid, a trait determinant, a gene or a marker is located. The loci of the present disclosure comprise one or more polymorphisms in a population; for example, alternative alleles are present in some individuals. As used herein, an "allele" refers to an alternative nucleic acid sequence at a specific locus. The length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele may occur on one chromosome, while a second allele occurs on a second homologous chromosome, for example, as a different chromosome of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA located on a single chromosome.

[0243] As used herein, centimorgan ("cM") is a unit of measurement for recombination frequency. 1 cM is equal to 1% chance that a marker at one locus will separate from a marker at a second locus due to hybridization in a single generation. Gene distances referred to herein can be calculated from recombination values ​​using the Kosambi function (Kosambi, The estimation of map distances from recombination values. Annals of Eugenics, 12: 172-75 (1944)).

[0244] As used herein, "tight linkage" or "association" means that a marker or locus is within about 20 cM, 10 cM, 5 cM, 1 cM, 0.5 cM or less than 0.5 cM of another marker or locus. For example, 20 cM means that the frequency of recombination between the marker and the locus is equal to or less than about 20%.

[0245] As used herein, "introgression" or "introgress" refers to the transmission of a desired allele of a genetic locus from one genetic background to another.

[0246] As used herein, "crossed" or "cross" means the production of offspring by fertilization (eg, cells, seeds, or plants), and includes crossing between plants (sexual) and self-fertilization (selfing).

[0247] As used herein, "backcrossing" refers to the process in which an offspring plant repeatedly crosses back to one of its parents. In a backcrossing scheme, a "donor" parent refers to a parent plant with a desired gene or locus to be infiltrated. A "recipient" parent (used once or multiple times) or a "recurrent" parent (used twice or multiple times) refers to a parent plant into which a gene or locus is infiltrated. The initial hybridization produces an F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is repeated, and the offspring individuals of each continuous backcross generation are themselves backcrossed to the same parental genotype.

[0248] As used herein, "monogenically transformed" or "monogenic transformation" refers to a plant developed using a plant breeding technique called backcrossing or by genetic engineering in which essentially all of the desired morphological and physiological characteristics of the variety are restored in addition to the single gene transferred into the variety by the backcrossing technique or by genetic engineering.

[0249] As used herein, "elite variety" means any variety resulting from breeding and selection with superior agronomic performance.

[0250] As used herein, "selecting" or "selection" in the context of marker-assisted selection or breeding refers to the act of picking or choosing desired individuals, usually from a population, based on certain predetermined criteria.

[0251] As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be affected by genotype. The phenotype can be observed with the naked eye, or by any other evaluation means known in the art, such as microscopy, biochemical analysis, genomic analysis, determination of tolerance to specific diseases, etc. In some cases, the phenotype is directly controlled by a single gene or locus, such as a "monogenic trait". In other cases, the phenotype is the result of several genes.

[0252] As used herein, "marker assay" means a method for detecting a polymorphism at a specific locus using a specific method, such as measuring at least one phenotype (such as seed color, flower color or other visually detectable traits), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based technology and nucleic acid sequencing technology, etc.

[0253] As used herein, "marker assisted selection" (MAS) is a method of selecting a phenotype based on a marker genotype. "Marker assisted selective breeding" refers to a method of selecting a desired trait in one or more plants by detecting one or more nucleic acids of a plant and then selecting plants or germplasm having the one or more nucleic acids, wherein the nucleic acid is linked to the desired trait.

[0254] As used herein, "polymorphism" refers to the presence of one or more changes in a population. Polymorphism can be manifested as changes in the nucleotide sequence of a nucleic acid or changes in the amino acid sequence of a protein. Polymorphism includes one or more changes in the nucleic acid sequence or nucleic acid characteristics of one or more loci in a population of one or more individuals. Changes may include, but are not limited to, one or more nucleotide base changes, the insertion of one or more nucleotides, or the deletion of one or more nucleotides. Polymorphism can be generated by random processes in nucleic acid replication, by mutagenesis, as a result of mobile genomic elements, by copy number changes and during meiosis, such as unequal hybridization, genome replication, and chromosome breakage and fusion. Changes are usually found in populations or can exist at low frequencies, the former having greater utility in general plant breeding, and the latter may be related to rare but important phenotypic changes. Useful polymorphisms may include single nucleotide polymorphisms (SNPs), insertions or deletions (Indels) in DNA sequences, simple sequence repeats (SSRs) of DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs. Genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, tolerance loci, satellite markers, transgenics, mRNAs, ds mRNAs, transcription profiles, and methylation patterns also include polymorphisms. In addition, the presence, absence, or variation of the aforementioned copy number may include polymorphisms.

[0255] As used herein, "SNP" or "single nucleotide polymorphism" refers to a sequence variation that occurs when a single nucleotide (A, T, C or G) in a genomic sequence is altered or variable. When a SNP is mapped to a site on a genome, a "SNP marker" is present.

[0256] As used herein, "marker" or "molecular marker" or "marker locus" is a term used to represent a nucleic acid or amino acid sequence that is unique enough to characterize a specific locus on a genome. Any detectable polymorphic trait can be used as a marker, as long as it is differentially inherited and exhibits linkage disequilibrium with the target phenotypic trait. Therefore, each marker is an indicator of a specific DNA fragment with a unique nucleotide sequence. The mapping position provides a measure of the relative position of a specific marker to each other. When describing a trait linked to a given marker, it should be understood that the actual DNA fragment whose sequence affects the trait is usually co-separated with the marker. If markers are identified on both sides of the trait, a more accurate and definite positioning of the trait can be obtained. By measuring the appearance of the marker in the hybrid offspring, the presence of the trait can be detected by relatively simple molecular tests without actually evaluating the appearance of the trait itself, which may be difficult and time-consuming, because the actual evaluation of the trait requires the plant to be grown to a stage and / or environmental conditions where the shape can be expressed. In one aspect, the marker used exhibits a LOD score of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more, measured using the Nic1b_ERF or Nic2_ERF locus using methods known in the art (such as Qgene Version 2.23 (1996)) and default parameters.

[0257] It is understood that any tobacco plant of the present disclosure may further comprise other agronomically desirable traits, such as by transformation with a genetic construct or transgene using techniques known in the art. Without limitation, examples of desirable traits are herbicide resistance, pest resistance, disease resistance; high yield; high grade index value; curability; ripening quality; mechanical harvestability; holding capacity; leaf quality; height, plant maturity (e.g., early, early mid, mid, mid-late, or late); stem size (e.g., small, medium, or large stem); or number of leaves per plant (e.g., small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21 leaves), or any combination. In one aspect, the disclosed reduced-nicotine or nicotine-free tobacco plants or seeds comprise one or more transgenes expressing one or more insecticidal proteins, e.g., crystal proteins of Bacillus thuringiensis or nutritional insecticidal proteins of Bacillus cereus, such as VIP3 (see, e.g., Estruch et al., 1996). al. (1997) Nat. Biotechnol. 15: 137). In another aspect, the tobacco plant further comprises an introgressed trait that confers resistance to brown stalk rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S. Pat. No. 5,491,081).

[0258] The disclosure also provides tobacco plant, it comprises the nicotine or total alkaloid level of change, but has the output suitable with the output of the corresponding initial tobacco plant that does not have such nicotine level change.In one aspect, low nicotine or nicotine-free tobacco variety provide the output selected from lower group: about 1200-3500,1300-3400,1400-3300,1500-3200,1600-3100,1700-3000,1800-2900,1900-2800,2000-2700,2100-2600,2200-2500 and 2300-2400lbs / acre. In another aspect, the reduced nicotine or nicotine-free tobacco variety provides a yield selected from the group consisting of about 1200-3500, 1300-3500, 1400-3500, 1500-3500, 1600-3500, 1700-3500, 1800-3500, 1900-3500, 2000-3500, 2100-3500, 2200-3500, 2300-3500, 2400-3500, 2500-3500, 2600-3500, 2700-3500, 2800-3500, 2900-3500, 3000-3500, and 3100-3500 lbs / acre. In another aspect, the reduced-nicotine or nicotine-free tobacco plant provides 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130% or 120%-130% of the yield of a control plant having essentially the same genetic background except for the nic1b_erf mutation, the nic2 mutation, the Nic1b_ERF transgenic, the Nic2 transgenic or a combination thereof. In another aspect, the reduced-nicotine or nicotine-free tobacco plant provides 70%-125%, 75%-120%, 80%-115%, 85%-110% or 90%-100% of the yield of a control plant having essentially the same genetic background except for the nic1b_erf mutation, the nic2 mutation, the Nic1b_ERF transgenic, the Nic2 transgenic, or a combination thereof.

[0259] In one aspect, disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) do not exhibit one or more, two or more, three or more or all of the LA BU21 traits selected from the group consisting of lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening. In one aspect, disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) do not exhibit two or more of the LA BU21 traits selected from the group consisting of lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening. In one aspect, disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) do not exhibit three or more LABU21 traits selected from the group consisting of: lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening. In one aspect, disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) exhibit lower levels of one or more, two or more, three or more or all of the LABU21 traits selected from the group consisting of: lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening compared to LABU21, LAFC53 or LNKY171. In one aspect, compared to LA BU21, LAFC53 or LNKY171, the disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) exhibit lower levels of two or more of the LA BU21 traits selected from the group consisting of lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening. In one aspect, compared to LA BU21, LAFC53 or LNKY171, the disclosed tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) exhibit lower levels of three or more or all of the LA BU21 traits selected from the group consisting of lower yield, delayed maturity and senescence, higher sensitivity to insect herbivory, increased polyamine content after topping, higher chlorophyll, more mesophyll cells per unit leaf area, and poorer end-product quality after ripening.

[0260] In one aspect, the disclosed modified tobacco plants (e.g., low nicotine, nicotine-free or low alkaloid tobacco varieties) comprise modifications that confer desired traits (e.g., low nicotine, nicotine-free or low alkaloids) without substantially affecting traits selected from the group consisting of yield, maturity and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll levels, number of mesophyll cells per unit leaf area, and end-product quality after ripening.

[0261] In one aspect, the disclosed modified tobacco plants comprise modifications that confer desired traits (e.g., low nicotine, nicotine-free, or low alkaloids), and also comprise traits that are substantially comparable to unmodified control plants, wherein the traits are selected from the group consisting of yield, maturity and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll levels, number of mesophyll cells per unit leaf area, and end-product quality after ripening.

[0262] In one aspect, disclosed modified tobacco plants comprise modifications that confer desired traits (e.g., low nicotine, nicotine-free or low alkaloids) and also comprise an output greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135% or greater than 140% relative to the output of unmodified control plants. In one aspect, disclosed modified tobacco plants comprise modifications that confer desired traits (e.g., low nicotine, nicotine-free or low alkaloids) and also comprise an output of 70%-140%, 75%-135%, 80%-130%, 85%-125%, 90%-120%, 95%-115% or 100%-110% relative to the output of unmodified control plants. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise a yield of 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125%-135%, or 130%-140% relative to the yield of unmodified control plants.

[0263] In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise a polyamine content after topping that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the polyamine content after topping of an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids), and further comprise a post-topping polyamine content of 70%-140%, 75%-135%, 80%-130%, 85%-125%, 90%-120%, 95%-115%, or 100%-110% relative to the post-topping polyamine content of an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise a post-topping polyamine content of 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125-135%, or 130%-140% relative to the post-topping polyamine content of an unmodified control plant.

[0264] In one aspect, the disclosed modified tobacco plant comprises a modification that imparts a desired trait (e.g., low nicotine, nicotine-free or low alkaloids), and further comprises a chlorophyll level that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135% or greater than 140% relative to the chlorophyll level of an unmodified control plant. In one aspect, the disclosed modified tobacco plant comprises a modification that imparts a desired trait (e.g., low nicotine, nicotine-free or low alkaloids), and further comprises a chlorophyll level that is greater than 70%-140%, 75%-135%, 80%-130%, 85%-125%, 90%-120%, 95%-115% or 100%-110% relative to the chlorophyll level of an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise chlorophyll levels of 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125%-135%, 130%-140% relative to the chlorophyll levels of unmodified control plants.

[0265] In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free or low alkaloids) and further comprise a mesophyll cell number per unit leaf area that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135% or greater than 140% relative to the number of mesophyll cells per unit leaf area of ​​an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids), and further comprise a mesophyll cell number per unit leaf area of ​​70%-140%, 75%-135%, 80%-130%, 85%-125%, 90%-120%, 95%-115%, or 100%-110% relative to the number of mesophyll cells per unit leaf area of ​​an unmodified control plant. In one aspect, the disclosed modified tobacco plants comprise modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids), and further comprise a mesophyll cell number per unit leaf area of ​​70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125%-135%, or 130%-140% relative to the number of mesophyll cells per unit leaf area of ​​an unmodified control plant.

[0266] In one aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are suitable for machine harvesting. In another aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are mechanically harvested.

[0267] In one aspect, the tobacco plant provided is a hybrid plant.Hybrid can be by stopping the self-pollination of the female parent plant (for example, seed parent) of the first variety, so that the pollen from the male parent plant of the second variety makes the female parent plant fertilized, and F1 hybrid seed is formed on the female plant to produce.The self-pollination of female plant can be stopped by flower emasculation in the early stage of flower development.Or, male sterile form can be used to stop the formation of pollen on the female parent plant.For example, male sterility can be produced by male sterility (MS) or transgenic male sterility, and wherein transgenic suppresses microspore formation and / or pollen formation, or self incompatibility.The parent plant containing MS is particularly useful.Aspects of female parent plant being MS, pollen can be gathered in the crops from male fertile plants and artificially applied to the style of MS female parent plant, and the results obtain F1 seed.

[0268] Plant can be used for forming single cross tobacco F1 hybrid.To be artificially transferred to the maternal plant of emasculation or the maternal plant of male sterility to form F1 seed from the pollen of male parent plant.Perhaps, can carry out three-way hybridization, wherein the single cross F1 hybrid is used as maternal and hybridizes with different male parents.As another selection, can create double cross hybrid, wherein the F1 offspring self hybridization of two different single crosses.When forming double cross hybrid, self-incompatibility can be used to prevent the self-pollination of maternal especially advantageously.

[0269] In one aspect, low nicotine or nicotine-free tobacco varieties are male sterile. On the other hand, low nicotine or nicotine-free tobacco varieties are cytoplasmic male sterile. Male sterile tobacco plants can be produced by any method known in the art. The method for producing male sterile tobacco plants is described in Wemsman, EA, and Rufty, RC1987.ChapterSeventeen.Tobacco.Pages 669-698 In:Cultivar Development.Crop Species.WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY761pp.

[0270] On the other hand, the tobacco part provided includes but is not limited to leaf, stem, root, seed, flower, pollen, anther, ovule, stalk node, fruit, meristem, cotyledon, hypocotyl, pod, embryo, endosperm, explant, callus, tissue culture, bud, cell and protoplast.In one aspect, the tobacco part provided does not comprise seed.In one aspect, the disclosure provides tobacco plant cell, tissue and organ, which are not propagation material and the natural reproduction of non-mediated plant.On the other hand, the disclosure also provides tobacco plant cell, tissue and organ, which are propagation material and the natural reproduction of mediated plant.On the other hand, the disclosure provides tobacco plant cell, tissue and organ that can not maintain self by photosynthesis.On the other hand, the disclosure provides somatic tobacco plant cell.Somatic cell (contrary to germline cell) does not mediate plant reproduction.

[0271] The cell, tissue and organ provided can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root, bud, stem, pod, flower, inflorescence, stalk, stalk, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue.On the other hand, the disclosure provides tobacco plant chloroplast.On the other hand, the disclosure provides epidermal cell, stomatal cell, leaf or root hair, storage root or tuber.On the other hand, the disclosure provides tobacco protoplast.

[0272] Those skilled in the art will appreciate that tobacco plants are naturally propagated by seeds, rather than by asexual reproduction or asexual reproduction. In one aspect, the disclosure provides tobacco endosperm. On the other hand, the disclosure provides tobacco endosperm cells. On the other hand, the disclosure provides male or female sterile tobacco plants that can not be bred without human intervention.

[0273] In one aspect, the disclosure provides a nucleic acid molecule comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-72, 147, 150, 157-161, 187-189 and 202-205, and fragments thereof. In one aspect, the present disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207 and 190-192. In another aspect, the present disclosure provides polypeptides or proteins comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207 and 190-192. In another aspect, the present disclosure provides biologically active variants of proteins having an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207 and 190-192. Biologically active variants of proteins disclosed herein may differ from the protein by as few as 1-15 amino acid residues, as few as 10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. Orthologous genes or proteins of genes or proteins from the Nic1b_ERF locus are also provided. "Orthologs" are genes derived from a common ancestral gene and are found in different species due to speciation. Orthologs may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity at the nucleotide sequence and / or protein sequence level. The functions of orthologs are generally highly conserved among species.

[0274] As used herein, the terms "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refer to the residues that are identical in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used with reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), and thus do not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitutions. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity."

[0275] The nucleic acid molecules, polypeptides or proteins provided can be isolated or substantially purified. An "isolated" or "purified" nucleic acid molecule, polypeptide, protein or biologically active portion thereof is substantially or substantially free of components that are normally associated with or interact with the polynucleotide or protein found in its naturally occurring environment. For example, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant technology, or is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0276] The disclosure also provides the method for making the tobacco product of the tobacco material that comprises disclosed tobacco plant.In one aspect, method comprises adjusting the aged tobacco material made by tobacco plant to increase its moisture content from about 12.5%-about 13.5% to about 21%, and the tobacco material of adjustment is blended to produce required admixture.In one aspect, the method for making tobacco product also comprises admixture casing or seasoning.Usually, in casing process, casing or sauce material are added in the admixture, to improve their quality and produce some required flavor characteristics by balancing chemical composition.At Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, more details of this casing method can be found in 1999.

[0277] The tobacco material provided can also be processed using methods including but not limited to heat treatment (for example, cooking, baking), seasoning, enzyme treatment, puffing and / or aging. These technical processing fermented and unfermented tobacco can be used. The example of suitable processed tobacco comprises dark air-cured tobacco, dark cured tobacco, burley tobacco, cured tobacco and cigar filling or packaging cigarette, and the products made by whole leaf stem processing. In one aspect, tobacco fiber comprises dark tobacco based on fresh weight at the most 70%. For example, tobacco can be regulated by heating, moisture removal and / or pasteurization steps described in U.S. Publication No. 2004 / 0118422 or 2005 / 0178398.

[0278] The tobacco material provided can be fermented. The characteristics of fermentation are typically high initial moisture content, heat production and 10-20% dry weight loss. See, for example, U.S. Patent Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition to modifying the fragrance of leaves, fermentation can change the color and / or texture of leaves. Also during fermentation, escaping gas can be produced, oxygen can be absorbed, pH can be changed, and the amount of water retained can be changed. See, for example, U.S. Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, or cured and fermented tobacco can be further processed (for example, shredded, expanded, blended, ground or pulverized) before being incorporated into oral products. In some cases, the tobacco is long cut fermented cured wet tobacco having an oven volatiles content of 48-50% by weight prior to mixing with the copolymer and optional flavorants and other additives.

[0279] In one aspect, the tobacco material provided can be processed into desired size. In one aspect, tobacco fiber is processed into the average fiber size less than 200 microns. In one aspect, tobacco fiber is 75-125 micron. In another aspect, tobacco fiber is processed into 75 microns or less size. In one aspect, tobacco fiber comprises long cut tobacco, which can be shredded or chopped into about 10 / inch to about 110 / inch width and about 0.1 inch to about 1 inch length. Double cut tobacco fiber can have a certain scope of particle size, makes about 70% double cut tobacco fiber between the mesh size of-20 mesh to 80 mesh.

[0280] The tobacco material provided can be processed to have an oven volatile content of about 10% by weight or more; about 20% by weight or more; about 40% by weight or more; about 15% by weight to about 25% by weight; about 20% by weight to about 30% by weight; about 30% by weight to about 50% by weight; about 45% by weight to about 65% by weight; or about 50% by weight to about 60% by weight. It will be understood by those skilled in the art that "wet" tobacco typically refers to tobacco having an oven volatile content of about 40% by weight to about 60% by weight (e.g., about 45% by weight to about 55% by weight, or about 50% by weight). As used herein, "oven volatiles" are determined by calculating the weight loss percentage of the sample after drying the sample in a preheated forced draft oven at 110°C for 3.25 hours. Oral products may have a total oven volatile content different from the oven volatile content of the tobacco fiber used to prepare the oral product. The described processing steps may reduce or increase the oven volatile content.

[0281] The following list provides a first set of exemplary embodiments.

[0282] 1. A tobacco plant or tobacco genotype or tobacco part thereof comprising a mutation in the Niclb locus, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 50 or greater after curing.

[0283] 2. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant further comprises a mutation in the ERF gene of the Nic2 locus.

[0284] 3. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more or all seven genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168.

[0285] 4. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant further comprises one or more mutations in ERF189, ERF115 or both.

[0286] 5. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value selected from the group consisting of 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, and 95 or higher.

[0287] 6. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value comparable to that of a control plant when grown and cured under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0288] 7. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value when cured that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% of the USDA Grade Index value of a control plant when grown and cured under similar conditions, wherein the control plant has essentially the same genetic background as the tobacco plant except for the mutation.

[0289] 8. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant comprises a nicotine level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level of a control plant when grown under similar growth conditions, wherein the control plant has essentially the same genetic background as the tobacco plant except for the mutation.

[0290] 9. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant comprises a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% and less than 0.05%.

[0291] 10. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 1, wherein the tobacco plant further comprises a transgene or mutation that directly inhibits the expression or activity of one or more genes encoding products selected from the group consisting of: PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP) and MATE transporter.

[0292] 11. A tobacco plant or tobacco genotype or tobacco part thereof comprising a mutation in the Nic1b locus, wherein the tobacco plant when cured is capable of producing a tobacco plant having a USDA grade index value comparable to a control plant when grown and cured under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0293] 12. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 11, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0294] 13. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 11, wherein the tobacco plant comprises one or more mutations in two or more, three or more, four or more, five or more, six or more or all seven genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17 and ERF168.

[0295] 14. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 11, wherein the tobacco plant further comprises one or more mutations in ERF189, ERF115 or both.

[0296] 15. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 11, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA Grade Index value selected from the group consisting of 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, and 95 or higher.

[0297] 16. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 11, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% of the grade index of a control plant.

[0298] 17. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 11, wherein the tobacco plant comprises a nicotine level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level of a control plant grown under similar growing conditions.

[0299] 18. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 11, wherein the tobacco plant further comprises a transgene or mutation that directly inhibits the expression or activity of one or more genes encoding products selected from the group consisting of: PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP) and MATE transporter.

[0300] 19. A plant or tobacco genotype of a tobacco variety comprising a nic1b mutation, wherein the tobacco variety has a leaf grade index that is comparable to the leaf grade index of a control tobacco variety when grown under similar growing conditions, wherein the control tobacco variety has substantially the same genetic background as the tobacco variety except for the mutation.

[0301] 20. The plant or tobacco genotype of embodiment 19, wherein the tobacco variety further comprises a mutation in the ERF gene at the Nic2 locus.

[0302] 21. The plant or tobacco genotype of embodiment 19, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of: ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0303] 22. The plant or tobacco genotype of embodiment 19, wherein the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both.

[0304] 23. A non-transgenic tobacco plant or tobacco genotype, or tobacco part thereof, comprising a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% and less than 0.05%, wherein the tobacco plant when cured is capable of producing leaves having a USDA Grade Index value of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more and 95 or more.

[0305] 24. The non-transgenic tobacco plant or tobacco genotype or tobacco part thereof of embodiment 23, wherein the non-transgenic tobacco plant comprises a nicotine level of less than 2.0% and is capable of producing leaves having a USDA Grade Index value of 70 or greater when cured.

[0306] 25. The non-transgenic tobacco plant or tobacco genotype or tobacco part thereof of embodiment 23, wherein the non-transgenic tobacco plant comprises a nicotine level of less than 1.0% and is capable of producing leaves having a USDA Grade Index value of 70 or greater when cured.

[0307] 26. A tobacco plant or tobacco genotype, or tobacco part thereof, comprising a non-transgenic mutation in the Niclb locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value comparable to the USDA Grade Index value of the control plant when cured, and wherein the control plant has essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

[0308] 27. A population of tobacco plants according to any one of embodiments 1-26.

[0309] 28. Cured tobacco material derived from the tobacco plant of any one of embodiments 1-26.

[0310] 29. The cured tobacco material of embodiment 28, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0311] 30. A tobacco blend comprising the cured tobacco material of embodiment 28.

[0312] 31. The tobacco blend of embodiment 30, wherein the cured tobacco material constitutes about at least 10 weight percent, at least 15 weight percent, at least 20 weight percent, at least 25 weight percent, at least 30 weight percent, at least 35 weight percent, at least 40 weight percent, at least 45 weight percent, at least 50 weight percent, at least 55 weight percent, at least 60 weight percent, at least 65 weight percent, at least 70 weight percent, at least 75 weight percent, at least 80 weight percent, at least 85 weight percent, at least 90 weight percent or at least 95 weight percent of the cured tobacco in the tobacco blend.

[0313] 32. The tobacco blend of embodiment 30, wherein the cured tobacco material constitutes about at least 10 volume %, at least 15 volume %, at least 20 volume %, at least 25 volume %, at least 30 volume %, at least 35 volume %, at least 40 volume %, at least 45 volume %, at least 50 volume %, at least 55 volume %, at least 60 volume %, at least 65 volume %, at least 70 volume %, at least 75 volume %, at least 80 volume %, at least 85 volume %, at least 90 volume % or at least 95 volume % of the cured tobacco in the tobacco blend.

[0314] 33. A tobacco product comprising the cured tobacco material of embodiment 28.

[0315] 34. The tobacco product of embodiment 33, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0316] 35. The tobacco product of embodiment 33, wherein the tobacco product is a smokeless tobacco product.

[0317] 36. The tobacco product of embodiment 35, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and snuff.

[0318] 37. Reconstituted tobacco comprising the cured tobacco material of embodiment 28.

[0319] 38. A tobacco plant or tobacco genotype or tobacco part thereof comprising a mutation in the Niclb locus, wherein the mutation is not present in the LA Burley 21 variety.

[0320] 39. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the tobacco plant comprises a shorter chromosome introgression at the Nic1b locus compared to the LA Burley 21 variety.

[0321] 40. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein said tobacco plant comprises a lower level of nicotine compared to a control tobacco plant not having said mutation when grown under similar growing conditions.

[0322] 41. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the tobacco plant comprises a nicotine level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level in a control tobacco plant not having the mutation grown under similar growing conditions.

[0323] 42. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein said tobacco plant comprises a lower level of total alkaloids compared to a control tobacco plant not having said mutation when grown under similar growth conditions.

[0324] 43. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the tobacco plant comprises a lower level of one or more alkaloids selected from the group consisting of nicotine, nornicotine, anabasine and anatabine compared to a control tobacco plant not having the mutation when grown under similar growing conditions.

[0325] 44. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the tobacco plant comprises similar levels of one or more compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoid, cembrenoid, sugar esters and reducing sugars as compared to a control tobacco plant not having the mutation when grown under similar growth conditions.

[0326] 45. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the mutation is homozygous.

[0327] 46. ​​The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the mutation is heterozygous.

[0328] 47. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the mutation is selected from the group consisting of a point mutation, a deletion, an insertion, a duplication and an inversion.

[0329] 48. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the mutation is introduced by a method selected from the group consisting of random mutagenesis and targeted mutagenesis.

[0330] 49. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 48, wherein the targeted mutagenesis is mediated by a meganuclease, a zinc finger nuclease, a TALEN or a CRISPR.

[0331] 50. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0332] 51. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203 and 184-186, and fragments thereof.

[0333] 52. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 38, wherein said mutation reduces the expression or activity of said gene.

[0334] 53. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof.

[0335] 54. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205 and 54, and fragments thereof.

[0336] 55. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 202-205, 54 and 72, and fragments thereof.

[0337] 56. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205 and 54, and fragments thereof.

[0338] 57. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene comprising a coding sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205 and 54, and fragments thereof.

[0339] 58. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 53, 204, 205 and 54, and fragments thereof.

[0340] 59. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 87, 88 and 89, and fragments thereof.

[0341] 60. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the plant further comprises reduced levels of mRNA, protein, or both encoding one or more genes selected from the group consisting of PMT, MPO, QPT, BBL, MATE, and A622, compared to a tobacco plant not having the mutation when grown under similar growth conditions.

[0342] 61. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the plant further comprises a transgene or mutation that inhibits the expression or activity of one or more genes encoding a product selected from the group consisting of: PMT, MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP) and MATE transporter.

[0343] 62. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein said tobacco plant is a hybrid.

[0344] 63. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the part is selected from the group consisting of leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, peduncles, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperms, explants, callus, tissue culture, shoots, cells, and protoplasts.

[0345] 64. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark-cured tobacco, Galpao tobacco, and Oriental tobacco.

[0346] 65. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the tobacco plant is from a variety selected from the group consisting of Burley, Maryland, and dark air-cured tobacco.

[0347] 66. A population of tobacco plants according to embodiment 38.

[0348] 67. Cured tobacco material derived from the tobacco plant of embodiment 38.

[0349] 68. The cured tobacco material of embodiment 67, wherein the cured tobacco material comprises a lower level of nicotine compared to cured tobacco material from a control tobacco plant lacking the mutation.

[0350] 69. The cured tobacco material of embodiment 67, wherein the tobacco plant comprises a nicotine level of 0.2% to 0.6%.

[0351] 70. The cured tobacco material of embodiment 67, wherein the tobacco plant comprises a nicotine level of 1.0% to 3.0%.

[0352] 71. The cured tobacco material according to embodiment 67, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0353] 72. A tobacco blend comprising the cured tobacco material of embodiment 67.

[0354] 73. A tobacco product comprising the cured tobacco material of embodiment 67.

[0355] 74. The tobacco product of embodiment 73, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0356] 75. The tobacco product of embodiment 73, wherein the tobacco product is a smokeless tobacco product.

[0357] 76. The tobacco product of embodiment 75, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and snuff.

[0358] 77. Reconstituted tobacco comprising the cured tobacco material of embodiment 67.

[0359] 78. A recombinant DNA construct comprising a promoter that is functional in tobacco cells and operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192, and fragments thereof.

[0360] 79. A tobacco plant or tobacco genotype or tobacco part thereof comprising the recombinant DNA construct of embodiment 78.

[0361] 80. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 79, wherein said tobacco plant comprises a higher level of nicotine compared to a control tobacco plant not containing said recombinant DNA construct.

[0362] 81. Cured tobacco material derived from the tobacco plant of embodiment 79.

[0363] 82. A tobacco product comprising the cured tobacco material of embodiment 81.

[0364] 83. A method for increasing nicotine levels in a tobacco plant, the method comprising transforming the tobacco plant with the recombinant DNA construct of embodiment 78.

[0365] 84. A recombinant DNA construct comprising a promoter that is functional in tobacco cells and operably linked to a polynucleotide encoding an RNA molecule, wherein the RNA molecule is capable of binding to an RNA encoding a polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-107, 148, 151, 180-183, 206, 207, and 190-192, and fragments thereof, and wherein the RNA molecule inhibits expression of the polypeptide.

[0366] 85. A tobacco plant or tobacco genotype or tobacco part thereof comprising the recombinant DNA construct of embodiment 84.

[0367] 86. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 85, wherein the RNA molecule is selected from the group consisting of microRNA, siRNA and trans-acting siRNA.

[0368] 87. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 85, wherein the polynucleotide encodes a double-stranded RNA.

[0369] 88. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 85, wherein said tobacco plant comprises a lower level of nicotine compared to a control tobacco plant not containing said recombinant DNA construct.

[0370] 89. Cured tobacco material derived from the tobacco plant of embodiment 85.

[0371] 90. A tobacco product comprising the cured tobacco material of embodiment 89.

[0372] 91. A method for reducing nicotine levels in a tobacco plant, the method comprising transforming the tobacco plant with the recombinant DNA construct of embodiment 84.

[0373] 92. A tobacco plant or tobacco genotype or tobacco part thereof comprising a heterologous expression cassette comprising a Nic1b inhibitory sequence of a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203 and 184-186, and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and wherein the inhibitory sequence is conjugated to at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, Fragments of 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides have at least 90% sequence identity: SEQ ID NO: 3-37, 146, 149, 152-156, 202, 203 and 184-186, and fragments thereof.

[0374] 93. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 92, wherein the Niclb inhibitory sequence is capable of being transcribed as an inhibitory polynucleotide selected from the group consisting of a single-stranded RNA polynucleotide, a double-stranded RNA polynucleotide, and a combination thereof.

[0375] 94. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 92, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter and a tissue-preferred promoter.

[0376] 95. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 92, wherein the promoter is a root-specific promoter.

[0377] 96. A tobacco plant or tobacco genotype or tobacco part thereof, comprising a heterologous expression cassette, the heterologous expression cassette comprising a Nic1b inhibitory sequence of a gene, the gene comprising a sequence selected from the group consisting of SEQ ID NO: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72 and fragments thereof, wherein the inhibitory sequence is operably linked to a promoter functional in a plant cell, and wherein the inhibitory sequence is coupled to at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 202-205 and 72 and fragments thereof 7, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 nucleotides having at least 90% sequence identity: SEQ ID NO: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof.

[0378] 97. A method for introgressing a low nicotine trait into a tobacco variety, the method comprising:

[0379] a. hybridizing a first tobacco variety comprising a low nicotine trait with a second tobacco variety not having the low nicotine trait to produce one or more offspring tobacco plants;

[0380] b. genotyping the one or more offspring tobacco plants for a polymorphic marker linked to a low nicotine trait, wherein the polymorphic marker is in a chromosomal interval flanked by any two of the SNP markers selected from the group consisting of: SEQ ID No. 125-145 and 193-201; and

[0381] c. Selecting progeny tobacco plants that contain the low nicotine trait.

[0382] 98. The method of embodiment 97, wherein the method further comprises backcrossing the selected progeny tobacco plant to the second tobacco variety.

[0383] 99. The method of embodiment 97, wherein the method further comprises:

[0384] d. crossing the selected progeny plant with itself or with a second tobacco variety to produce one or more further progeny tobacco plants; and

[0385] e. Selecting another progeny tobacco plant comprising the low nicotine trait.

[0386] 100. The method of embodiment 99, wherein the selection step (e) comprises marker-assisted selection.

[0387] 101. The method of embodiment 97, wherein the method produces a single gene transformation comprising a low nicotine trait.

[0388] 102. The method of embodiment 97, wherein the second tobacco variety is an elite variety.

[0389] 103. The method of embodiment 97, wherein the genotyping involves one or more molecular marker measurements.

[0390] 104. The method of embodiment 97, wherein the polymorphic marker comprises a polymorphism selected from the following groups: single nucleotide polymorphism (SNP), insertion or deletion (Indel) in DNA sequence, simple sequence repeat (SSR) of DNA sequence, restriction fragment length polymorphism (RFLP) and tag SNP.

[0391] 105. The method of embodiment 97, wherein the genotyping comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203, and 184-186, and fragments thereof.

[0392] 106. The method of embodiment 97, wherein the genotyping comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72 and fragments thereof.

[0393] 107. The method of embodiment 97, wherein the genotyping includes determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205 and 54 and fragments thereof.

[0394] 108. The method of embodiment 97, wherein the first tobacco variety is selected from the group consisting of LA Burley 21, LAFC53, and LNKY171.

[0395] 109. The method of embodiment 97, wherein the selected progeny tobacco plant comprises a shorter chromosome introgression at the Niclb locus compared to LA Burley 21, LAFC53, and LNKY171.

[0396] 110. A method for introgressing a low nicotine trait into a tobacco variety, the method comprising:

[0397] a. hybridizing a first tobacco variety comprising a low nicotine trait with a second tobacco variety not having the low nicotine trait to produce one or more offspring tobacco plants;

[0398] b. Genotyping the one or more offspring tobacco plants for a polymorphic marker linked to a low nicotine trait, wherein the polymorphic marker is within 20 cM of any one of the SNP markers selected from the group consisting of SEQ ID Nos. 125-145 and 193-201, or within any locus having a sequence selected from the group consisting of SEQ ID Nos. 1, 3-37, 146, 149, 152-156, 202, 203, and 184-186; and

[0399] c. Selecting progeny tobacco plants that contain the low nicotine trait.

[0400] 111. The method of embodiment 110, wherein the genotyping comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3-37, 146, 149, 152-156, 202, 203 and 184-186, and fragments thereof.

[0401] 112. The method of embodiment 110, wherein the genotyping comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof.

[0402] 113. The method of embodiment 110, wherein the genotyping comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205 and 54, and fragments thereof.

[0403] 114. A method for selecting a tobacco plant having a low nicotine trait, the method comprising:

[0404] a. Isolating nucleic acids from a collection of tobacco germplasm;

[0405] b. determining one or more markers in the nucleic acid that are closely linked to the Nic1b locus; and

[0406] c. Selecting tobacco plants having a low nicotine trait based on the marker assay.

[0407] 115. The method described in embodiment 114, wherein the one or more markers are within approximately 20cM, 10cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.5cM or less than 0.5cM of any one of the SNP markers selected from the following group: SEQ ID No.125-145 and 193-201, or within any locus having a sequence selected from the following group: SEQ ID No.1, 3-37, 146, 149, 152-156, 202, 203 and 184-186.

[0408] 116. The method described in embodiment 114, wherein the determination comprises determining a nucleic acid sequence located within a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity with a sequence selected from the following group: SEQ ID NO: 3-37, 146, 149, 152-156, 202, 203 and 184-186, and fragments thereof.

[0409] 117. The method of embodiment 114, wherein the determination comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 15, 17, 18, 19, 37, 39, 49, 50, 52, 53, 54, 202-205 and 72, and fragments thereof.

[0410] 118. The method of embodiment 114, wherein the determination comprises determining a nucleic acid sequence located within a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 14, 17, 18, 19, 49, 52, 53, 202-205 and 54, and fragments thereof.

[0411] 119. The method of embodiment 114, wherein the method further comprises measuring the nicotine level of the selected plant to determine the low-nicotine trait.

[0412] 120. The method of embodiment 114, wherein the collection of tobacco germplasm is a haploid breeding population.

[0413] 121. A tobacco plant or tobacco genotype or tobacco part thereof, comprising a chromosomal introgression obtainable from any one of LA Burley 21, LA FC53 and LN KY171, and flanked by and not comprising any two of the SNP markers selected from the group consisting of SEQ ID Nos. 125-145 and 193-201, or flanked by any two loci of sequences selected from the group consisting of SEQ ID Nos. 1, 3-37, 146, 149, 152-156, 202, 203 and 184-186, wherein the tobacco plant is capable of producing leaves having a USDA grade index value of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, or 95 or more when mature.

[0414] 122. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 121, wherein the tobacco plant comprises a nicotine level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% and less than 0.05%.

[0415] 123. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 121, wherein the chromosomal introgression is flanked by and does not comprise any two of the SNP markers selected from the group consisting of SEQ ID Nos. 126-135.

[0416] 124. The tobacco plant or tobacco genotype or tobacco part thereof according to embodiment 121, wherein the chromosomal introgression is flanked by and does not comprise the SNP markers SEQ ID Nos. 129-132.

[0417] 125. A tobacco plant or tobacco genotype or tobacco part thereof comprising a first chromosome introgression obtainable from any one of LA Burley 21, LAFC53 and LNKY171, and flanked by and not comprising any two of Nic1b markers No. 1-207, wherein the tobacco plant when mature is capable of producing leaves having a USDA grade index value comparable to a control plant grown under similar growth conditions, wherein the control plant has essentially the same genetic background as the tobacco plant except for the mutation.

[0418] 126. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 125, wherein the tobacco plant comprises a nicotine level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70% or less than 80% of the nicotine level of a control plant grown under similar growing conditions.

[0419] 127. A population of tobacco plants according to any one of embodiments 121-126 and 136-141.

[0420] 128. Cured tobacco material derived from the tobacco plant of any one of embodiments 121-126.

[0421] 129. The cured tobacco material of embodiment 128, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0422] 130. A tobacco blend comprising the cured tobacco material of embodiment 128.

[0423] 131. The tobacco blend of embodiment 130, wherein the cured tobacco material constitutes about at least 10 weight percent, at least 15 weight percent, at least 20 weight percent, at least 25 weight percent, at least 30 weight percent, at least 35 weight percent, at least 40 weight percent, at least 45 weight percent, at least 50 weight percent, at least 55 weight percent, at least 60 weight percent, at least 65 weight percent, at least 70 weight percent, at least 75 weight percent, at least 80 weight percent, at least 85 weight percent, at least 90 weight percent, or at least 95 weight percent of the cured tobacco in the tobacco blend.

[0424] 132. The tobacco blend of embodiment 130, wherein the cured tobacco material constitutes about at least 10 volume %, at least 15 volume %, at least 20 volume %, at least 25 volume %, at least 30 volume %, at least 35 volume %, at least 40 volume %, at least 45 volume %, at least 50 volume %, at least 55 volume %, at least 60 volume %, at least 65 volume %, at least 70 volume %, at least 75 volume %, at least 80 volume %, at least 85 volume %, at least 90 volume % or at least 95 volume % of the cured tobacco in the tobacco blend.

[0425] 133. A tobacco product comprising the cured tobacco material of embodiment 128.

[0426] 134. The tobacco product of embodiment 133, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0427] 135. The tobacco product of embodiment 133, wherein the tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and snuff.

[0428] 136. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 146, 153, 154, 17, 18, 19, 202 and 203, and fragments thereof.

[0429] 137. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 153, 154, 17, 18, 19, 202 and 203, and fragments thereof.

[0430] 138. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 153, 154, 17, 18, 19 and 203, and fragments thereof.

[0431] 139. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 17, 18 and 19, and fragments thereof.

[0432] 140. The tobacco plant or part thereof of embodiment 38, wherein the mutation is within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 18 and 19, and fragments thereof.

[0433] 141. The tobacco plant or tobacco genotype or tobacco part thereof of embodiment 38, wherein the mutation is located in a gene encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206 and 207, and fragments thereof.

[0434] The following list provides a second set of exemplary embodiments.

[0435] 1. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 50 or greater when cured.

[0436] 2. The tobacco plant or part thereof of embodiment 1, wherein the tobacco plant is a Nicotiana tabacum plant.

[0437] 3. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0438] 4. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0439] 5. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 70 or greater when cured.

[0440] 6. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value comparable to that of a control plant when grown and cured under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0441] 7. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant comprises a nicotine level that is less than 40% of the nicotine level of a control plant when grown under similar growing conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0442] 8. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant comprises a nicotine level of less than 2.0%.

[0443] 9. A tobacco plant or part thereof comprising a non-transgenic mutation in the Nic1b_ERF locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 60% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves having a USDA grade index value comparable to the USDA grade index value of the control plant when cured, and wherein the control plant has essentially the same genetic background as the tobacco plant except for the non-transgenic mutation, wherein the tobacco plant is a Nicotiana tabacum plant.

[0444] 10. A population of tobacco plants according to embodiment 2.

[0445] 11. Cured tobacco material derived from the tobacco plant of embodiment 2.

[0446] 12. The cured tobacco material of embodiment 11, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0447] 13. A tobacco blend comprising the cured tobacco material of embodiment 11.

[0448] 14. A tobacco product comprising the cured tobacco material of embodiment 11.

[0449] 15. The tobacco product of embodiment 14, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0450] 16. The tobacco product of embodiment 14, wherein the tobacco product is a smokeless tobacco product.

[0451] 17. The tobacco product of embodiment 16, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and snuff.

[0452] 18. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the mutation is not present in LABurley 21, LA FC53 and LN KY171.

[0453] 19. The tobacco plant or part thereof of embodiment 18, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0454] 20. The tobacco plant or part thereof of embodiment 18, wherein the mutation is within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54 and 202-205, and fragments thereof.

[0455] 21. The tobacco plant or part thereof of embodiment 18, wherein the mutation is in a gene encoding a polypeptide having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206 and 207.

[0456] 22. The tobacco plant or part thereof of embodiment 18, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark-cured tobacco, Galpao tobacco, and Oriental tobacco.

[0457] 23. The tobacco plant or portion thereof of embodiment 18, wherein the tobacco plant is from a variety selected from the group consisting of Burley, Maryland, and dark air-cured tobacco.

[0458] 24. Cured tobacco material derived from the tobacco plant of embodiment 18.

[0459] 25. The cured tobacco material of embodiment 24, wherein the tobacco plant comprises a nicotine level of 0.2% to 0.6%.

[0460] 26. The cured tobacco material of embodiment 24, wherein the tobacco plant comprises a nicotine level of 1.0% to 3.0%.

[0461] 27. The cured tobacco material of embodiment 24, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0462] 28. A tobacco product comprising the cured tobacco material of embodiment 24.

[0463] 29. The tobacco product of embodiment 24, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0464] 30. The tobacco product of embodiment 24, wherein the tobacco product is a smokeless tobacco product.

[0465] A third set of exemplary embodiments is provided below:

[0466] 1. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 50 or greater when cured.

[0467] 2. The tobacco plant or part thereof of embodiment 1, wherein the tobacco plant is a Nicotiana tabacum plant.

[0468] 3. The tobacco plant or part thereof of embodiment 1 or 2, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0469] 4. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the ERF gene of the Nic2 locus is NCG1.

[0470] 5. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the ERF gene of the Nic2 locus is ERF16.

[0471] 6. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the ERF gene of the Nic2 locus is NCG15.

[0472] 7. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the ERF gene of the Nic2 locus is NCG17.

[0473] 8. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the ERF gene of the Nic2 locus is ERF130.

[0474] 9. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0475] 10. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant is capable of producing leaves having a USDA Grade Index value of 70 or greater when cured.

[0476] 11. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant, when cured, is capable of producing leaves having a USDA grade index value comparable to that of a control plant when grown and cured under similar conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0477] 12. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant comprises a nicotine level that is less than 40% of the nicotine level of a control plant when grown under similar growing conditions, wherein the control plant has substantially the same genetic background as the tobacco plant except for the mutation.

[0478] 13. The tobacco plant or part thereof of embodiment 2, wherein the tobacco plant comprises a nicotine level of less than 2.0%.

[0479] 14. A tobacco plant or part thereof comprising a non-transgenic mutation in the Nic1b_ERF locus, wherein the non-transgenic mutation reduces the nicotine level of the tobacco plant to less than 60% of the nicotine level of a control plant when grown under similar growing conditions, wherein the tobacco plant is capable of producing leaves having a USDA grade index value comparable to the USDA grade index value of the control plant when cured, and wherein the control plant has essentially the same genetic background as the tobacco plant except for the non-transgenic mutation, wherein the tobacco plant is a Nicotiana tabacum plant.

[0480] 15. A population of tobacco plants according to embodiment 2 or 14.

[0481] 16. Cured tobacco material derived from the tobacco plant of embodiment 2 or 14.

[0482] 17. The cured tobacco material of embodiment 16, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0483] 18. A tobacco blend comprising the cured tobacco material of embodiment 16.

[0484] 19. A tobacco product comprising the cured tobacco material of embodiment 16.

[0485] 20. The tobacco product of embodiment 19, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0486] 21. The tobacco product of embodiment 19, wherein the tobacco product is a smokeless tobacco product.

[0487] 22. The tobacco product of embodiment 21, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and snuff.

[0488] 23. A tobacco plant or part thereof comprising a mutation in the Nic1b_ERF locus, wherein the mutation is not present in LABurley 21, LA FC53 and LN KY171.

[0489] 24. The tobacco plant or part thereof of embodiment 23, wherein the tobacco plant further comprises a mutation in the ERF gene at the Nic2 locus.

[0490] 25. The tobacco plant or part thereof of embodiment 23 or 24, wherein the ERF gene of the Nic2 locus is NCG1.

[0491] 26. The tobacco plant or part thereof according to any one of embodiments 23-25, wherein the ERF gene of the Nic2 locus is ERF16.

[0492] 27. The tobacco plant or part thereof according to any one of embodiments 23-26, wherein the ERF gene of the Nic2 locus is NCG15.

[0493] 28. The tobacco plant or part thereof according to any one of embodiments 23-27, wherein the ERF gene of the Nic2 locus is NCG17.

[0494] 29. The tobacco plant or part thereof according to any one of embodiments 23-28, wherein the ERF gene of the Nic2 locus is ERF130.

[0495] 30. The tobacco plant or part thereof of any one of embodiments 23-29, wherein the mutation is within a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17, 18, 19, 38, 48, 49, 52, 53, 54 and 202-205, and fragments thereof.

[0496] 31. The tobacco plant or part thereof of any one of embodiments 23-30, wherein the mutation is in a gene encoding a polypeptide having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87, 88, 89, 180, 181, 206 and 207.

[0497] 32. The tobacco plant or part thereof of any one of embodiments 23-31, wherein the tobacco plant is from a variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark-cured tobacco, Galpao tobacco, and Oriental tobacco.

[0498] 33. The tobacco plant, or portion thereof, of any one of embodiments 23-32, wherein the tobacco plant is from a variety selected from the group consisting of Burley, Maryland, and dark air-cured tobacco.

[0499] 34. Cured tobacco material derived from the tobacco plant of any one of embodiments 23-33.

[0500] 35. The cured tobacco material of embodiment 34, wherein the tobacco plant comprises a nicotine level of 0.2% to 0.6%.

[0501] 36. The cured tobacco material of embodiment 34, wherein the tobacco plant comprises a nicotine level of 1.0% to 3.0%.

[0502] 37. The cured tobacco material of embodiment 34, wherein the cured tobacco material is prepared by a curing method selected from the group consisting of flue-curing, air-curing, fire-curing and sun-curing.

[0503] 38. A tobacco product comprising the cured tobacco material of embodiment 34.

[0504] 39. The tobacco product of embodiment 34, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, rolling tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and shredded tobacco.

[0505] 40. The tobacco product of embodiment 34, wherein the tobacco product is a smokeless tobacco product.

[0506] 41. A modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having at least 90% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

[0507] 42. The modified tobacco plant or part thereof of embodiment 41, wherein the polynucleotide has at least 95% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

[0508] 43. The modified tobacco plant or part thereof of embodiment 41 or 42, wherein the polynucleotide has 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

[0509] 44. The modified tobacco plant, or part thereof, of any one of embodiments 41-43, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation.

[0510] 45. The modified tobacco plant or part thereof according to any one of embodiments 41-44, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

[0511] 46. ​​A modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having a nucleic acid sequence encoding a polypeptide having at least 90% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0512] 47. The modified tobacco plant or part thereof of embodiment 46, wherein the polynucleotide has a nucleic acid sequence encoding a polypeptide having at least 95% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0513] 48. The modified tobacco plant or part thereof of embodiment 46 or 47, wherein the polynucleotide has a nucleic acid sequence encoding a polypeptide having at least 100% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

[0514] 49. The modified tobacco plant or part thereof of any one of embodiments 46-48, wherein the modified tobacco plant comprises a reduced level of at least one alkaloid compared to a control tobacco plant lacking the mutation, relative to a control plant not having the genetic modification or mutation or recombinant nucleic acid construct.

[0515] 50. The modified tobacco plant, or part thereof, of any one of embodiments 46-49, wherein said higher USDA Grade Index value is at least 5% higher than said comparable leaves of said control plant.

[0516] 51. The tobacco plant or part thereof according to any one of the preceding embodiments, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine and nornicotine.

[0517] 52. Cured tobacco material derived from the tobacco plant of any one of the preceding embodiments.

[0518] 53. A tobacco product comprising the cured tobacco material of embodiment 52.

[0519] 54. The tobacco product of embodiment 53, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recessed filter cigarettes, ventilated recessed filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, cigarettes, chewing tobacco, leaf tobacco, cut and shredded tobacco, reconstituted tobacco, loose leaf chewing tobacco, plugged chewing tobacco, moist snuff, snuff and dry snuff, or smokeless tobacco products.

[0520] 55. A method for producing a modified tobacco plant, comprising:

[0521] (a) inducing a non-natural mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence that is at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207;

[0522] (b) selecting at least one tobacco cell from step (a) that comprises the non-natural mutation; and

[0523] (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b);

[0524] wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid as compared to a control tobacco plant lacking the mutation.

[0525] 56. The method of embodiment 55, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation.

[0526] 57. The method of any one of embodiments 55 or 56, wherein the endogenous nucleic acid sequence has at least 90% identity with a sequence selected from the following group: SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

[0527] 58. The method of any one of embodiments 55-57, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

[0528] 59. The method according to any one of embodiments 55-58, wherein the method further comprises:

[0529] (d) planting the modified tobacco plant regenerated in step (c).

[0530] 60. The method according to any one of embodiments 55-59, wherein the method further comprises:

[0531] (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and

[0532] (f) obtaining at least one seed from said crossing in step (e).

[0533] 61. The modified tobacco plant or part thereof of any one of embodiments 1-60, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation.

[0534] 62. The modified tobacco plant or part thereof according to embodiment 61, wherein the at least one alkaloid is anabasine.

[0535] 63. The modified tobacco plant or part thereof of embodiment 61 or 62, wherein the at least one alkaloid is anatabine.

[0536] 64. The modified tobacco plant or part thereof of embodiment 61, 62 or 63, wherein the at least one alkaloid is nicotine.

[0537] 65. The modified tobacco plant or part thereof of embodiment 61, 62, 63 or 64, wherein the at least one alkaloid is nornicotine.

[0538] Having generally described the disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure unless otherwise specified. DETAILED DESCRIPTION

[0539] Example 1: Development of TN90 lines with low alkaloid traits and plants with high and medium alkaloid traits comprising nic1 and nic2 deletion marker alleles

[0540] A breeding population was developed from a cross between LA BU21 (low alkaloid Burley 21 containing nic1 and nic2 mutations, donor parent) and elite cultivar TN90 (recipient parent). The aim was to obtain low alkaloid plants that also had improved leaf quality compared to the parental LA BU21 line with inferior leaf characteristics. The F2 plants with the desired characteristics (low alkaloid levels, improved leaf quality) were further selfed to obtain the F3 generation and subsequently the F5 generation. Figure 1 The breeding process used to develop an inbred TN90 line with a low alkaloid trait is shown.

[0541] like Figure 1 As shown, in 22 F3 plants were screened on the array. The array was developed based on approximately 170,000 polymorphic SNPs observed in several resequenced lines belonging to different tobacco types (Flue, Dark, Oriental and Burley) relative to the TN90 tobacco genome sequence database. Plant 22-6 showed approximately 40.6% recovery of the recipient parent genome and was selected for further improvement. The F3 generation also showed low alkaloid levels, consistent with those observed in LA BU21. The F4 generation was screened using the Nic1 and Nic2 KASP assays (see SEQ ID NOs: 135 and 137 of US2016 / 0374387, corresponding to Nic1, respectively). KASP and Nic2 KASP All 192 F4 plants screened had both deletions, and three plants (ds1059-138, ds1059-143, and ds1059-192) were selected for further improvement. Selfed seeds from these three plants were further expanded and evaluated in the F5 generation. All 220 F5 plants were positive for nic1 KASP and nic2 KASPThe deletion alleles were all homozygous. Alkaloid levels in F5 plants were determined by measuring the 3rd, 4th and 5th leaves from the top of the plant and the combined leaf samples collected 2 weeks after topping. Not all plants showed the low alkaloid trait, and significant differences in leaf phenotype were also observed, with several lines showing good leaf quality consistent with that observed in commercial burley tobacco (Table 1).

[0542] Table 1: Alkaloid levels and leaf phenotypes of selected F5 and control plants. All F5 plants were positive for nic1 KASP and nic2 KASP All deletion alleles were homozygous.

[0543]

[0544] Example 2: Identification of other genomic regions associated with low alkaloid traits

[0545] The F5 plants of Example 1 had high alkaloid levels despite carrying homozygous nic1 KASP and nic2 KASP Deletion allele, indicating that nic1 KASP and nic2 KASP The deletion markers did not show 100% linkage to the low alkaloid trait. To investigate this observation, LAFC53 (a bakery-ripened variety carrying the introgression of nic1 and nic2), LN KY171 (a dark variety carrying the introgression of nic1 and nic2), and KY171 (a dark variety without the introgression of nic1 and nic2), as well as the nic1 KASP and nic2 KASP Two (2) F5 lines (G61-36 and G61-39) that were homozygous for the deletion allele but showed high alkaloid levels, and KASP and nic2 KASP Two (2) F5 lines (G61-31 and G61-37) that were homogeneous for the deletion allele and exhibited low alkaloid levels were further resequenced.In addition to the previously resequenced lines, the following seven lines: BU21, HI BU21, LIBU21 and LA BU21 were mapped using the TN90 genome database.

[0546] Coverage analysis of the mapped reads led to the identification of a 2000-3000 bp region (SEQ ID No. 1, hereinafter referred to as "LA_associated_region"), approximately 2 million bp downstream of the nic1 deletion fragment NT1.0-Scaffold0002504 previously described in US2016 / 0374387. The LA_associated_region was found in LA BU21, LI BU21, LN KY171, LAFC53, and low alkaloid homozygous nic1 KASP nic2 KASP In contrast, LA_associated_region is absent in wild-type (e.g., TN90, BU21, K326, NLM, Katerini) and high alkaloid homozygous nic1 KASP nic2 KASP There is no deletion (i.e., presence) in the F5 strain, G61-36, and G61-39. There is no known gene in the LA_associated_region. A serine-threonine protein phosphatase gene is present 4877 bp upstream of this region, and an ERF (homolog of ERF189) is present 59,945 bp downstream of this region.

[0547] In addition, SNP genotyping of F3 plants (22-6) revealed that a genomic region starting from 1,256,063 bp and ending at 2,766,118 bp (about 1,510,055 bp long, SEQ ID No. 2, referred to herein as "Nic1b region") downstream of the nic1 deletion segment NT1.0-Scaffold0002504 was heterozygous in F3 plants (including LA_associated_region). This suggests that the low alkaloid allele in the Nic1b region may be derived from cigars (LA BU21 was produced by a cross between BU21 and a low alkaloid Cuban cigar variety) and segregated in the resulting F4 generation and subsequent generations.

[0548] In addition, 47 F4 plants and 48 F5 plants were genotyped using the Axiome array containing approximately 170,000 SNPs to further verify the correlation between alkaloid levels and the LA_associated_region and Nic1b interval. Analysis of three ancestral F4 plants (ds1059-138, ds1059-143, and ds1059-192) showed that the LA_associated_region and Nic1b regions were heterozygous or carried the LA BU21-like (B) allele. Similarly, 48 F5 plants showed segregation in LA_associated_region and Nic1b region, with individuals with the B allele (LA BU21-like) showing low alkaloid levels (0.137%-0.417%, dry weight), and individuals with the heterozygous state (H) or wild-type allele (A) showing moderate to high alkaloid levels (1.793%-5.419%, dry weight).

[0549] Example 3: Identification of genes and markers in the Nic1b region

[0550] Sequence analysis of the Nic1b region revealed at least 35 annotated genes from this region (Table 2). These genes are referred to as N ic1b cigar( C igar) gene G ene) ("NCG") 1-35. Among these NCG genes, six ethylene-responsive transcription factor-like (ERF-like) genes were identified. RNA-seq data showed that 10 NCGs showed reduced expression in LA BU21 plants relative to BU21 from root samples collected 72 hours after topping. Seven NCGs (including four of the six ERF-like genes) were downregulated in LA BU21 compared with BU21, while the other three NCGs were upregulated.

[0551] Each of the 35 NCGs in LA BU21, TN90, and selected F5 plants was resequenced to identify specific natural mutations present in LA BU21 that may cause loss of gene function. Additional resequencing analyses were also performed to identify mutations in non-coding sequences (e.g., promoters) that may cause dysregulated gene expression.

[0552] In addition, polymorphisms in and on both sides of the Nic1b region were identified to develop SNP markers. 21 SNP markers were developed for genotyping in the Nic1b region (Table 3). Other SNP markers are provided in Table 4. These markers are located in the Nic1b region and are adjacent to various ERF genes. They have alleles that can be used to distinguish normal alkaloid strains from low alkaloid strains.

[0553] Table 2: Annotated genes in the Nic1b region. a g31431 and g31432 represent alternative splicing (see Figure 2 ). b g31446 may represent another alternative splicing.

[0554]

[0555]

[0556] Table 3: Markers in and around the Nic1b region. (*) Genomic positions are based on the TN90 genome, which is also used to indicate the NCG gene positions.

[0557]

[0558]

[0559] Table 4: SNP markers in close proximity to multiple ERF genes from the Nic1b region. (*) Genomic positions are based on the reference TN90 genome.

[0560]

[0561]

[0562] Example 4: Transgenic methods for developing tobacco varieties with desired nicotine levels.

[0563] Overexpression and inhibition methods were used to study the function of NCGs and develop tobacco varieties with desired nicotine levels. Two groups of transgenic plants were produced, one group using an overexpression method with full-length coding sequences and the other using an inhibition method with artificial microRNA or RNAi sequences. All 35 NCGs (NCG1-NCG35) were tested, with emphasis on all NCGs showing differential expression between TN90 and LA BU21, especially those encoding ERF-like proteins. Some overexpression studies were performed in a nic1 nic2 double mutant background (e.g., LA BU21) to test the ability of the identified genes (alone or in combination) to complement or rescue the low alkaloid mutant phenotype from the mutant nic1 allele.

[0564] To express the full-length coding sequence or artificial microRNA sequence, an expression vector with a CsVMV promoter and a NOS terminator was constructed, as well as an expression cassette with a kanamycin selection marker (NPT II) under the direction of the actin 2 promoter and a NOS terminator. Exemplary artificial microRNA sequences can be found in Table 2. One of ordinary skill in the art will appreciate that other target sequences can be used to construct suppression constructs or other transgenic methods for gene silencing (e.g., RNAi, trans-acting siRNA, etc.).

[0565] Nucleic acid constructs carrying target transgenes were introduced into tobacco leaf discs using DNA bombardment or gene gun approaches. See, e.g., Sanford et al., 1993, Methods Enzymol., 217: 483-510; and Okuzaki and Tabei, 2012, Plant Biotechnology, 29: 307-310. Briefly, plasmid DNA containing the transformation cassette was coated on 1 μm gold particles (DNA / gold) as follows. The 1 μm gold particles were baked at 180°C for 12 hours, and a stock solution (40 mg / ml) was prepared. To prepare a mixture for 10 injections, 100 μl of the stock solution was mixed with 40 μl of expression vector DNA (1 μg / μl), 100 μl of 2.5 M CaCl2, and 40 μl of 0.1 M spermidine in a 1.5 ml tube. The mixture was centrifuged at 13,000 × g for 30 s, and the precipitate was washed with 500 μl of 100% ethanol. The DNA / gold mixture was suspended in 100 μl of water and 10 μl was added to the macrocarrier, dried, and then bombarded. In a PDS-1000 / He system (Bio-Rad Laboratories, Hercules, CA, USA), a 1,100 psi rupture disk was used to bombard each plate twice under partial vacuum (711 mmHg). Narrow Leaf Madole (NLM) and Tennessee 90 (TN 90) tobacco leaf discs were used to transform with RNAi constructs and full-length gene constructs. Whole tobacco leaves (approximately 45 × 30 mm) were placed on MS medium overnight and the leaf discs were bombarded with the constructs the next day. Then, the leaves are cut into small pieces (about 5 × 5mm) and put back into TOM medium (containing 20g sucrose / L; 1mg / L IAA and 2.5mg / L BAP MS medium), grown at 27°C for 3-5 days, then transferred to the TOM medium containing 300mg / l kanamycin (TOM-Kan) and grown. Every 2-3 weeks, the tissue is transferred to a new TOM-Kan plate for 4-6 weeks (27°C, 16h illumination). Kanamycin-resistant young shoots are regenerated 4-6 weeks after bombardment. Buds are transferred to MS-kanamycin flat plates and roots are grown. Then, leaves and / or roots from T1 plants (and subsequent generations) are evaluated to determine the amount of one or more alkaloids.

[0566] Example 5: Random mutagenesis to generate new mutations conferring low alkaloid traits in or around the Nic1b region

[0567] Random mutagenesis of tobacco plants was performed using ethyl methanesulfonate (EMS) mutagenesis or fast neutron bombardment. EMS mutagenesis consists of chemically induced random point mutations over the genome length. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment, which causes large deletions through double-strand DNA breaks.

[0568] For EMS mutagenesis, 1 g (about 10,000 seeds) of Tennessee 90 tobacco (TN90) seeds were washed in 0.1% Tween for 15 minutes and then soaked in 30 ml ddH2O for 2 hours. Then, one hundred and fifty (150) μl of 0.5% EMS (Sigma, catalog number M-0880) was mixed into the seeds / ddH2O solution and incubated for 8-12 h (30 rpm rotation) under the hood at room temperature (RT; about 20°C). Then, liquid was removed from the seeds and mixed into 1M sodium hydroxide overnight to purify and process. Then, seeds were washed with 100 ml ddH2O for 2-4 hours. Then, the washed seeds were suspended in 0.1% agar solution.

[0569] The EMS-treated seeds in the agar solution were evenly spread on the water-soaked Carolina's Choice Tobacco Mix (Carolina Soil Company, Kinston, NC) in the plates at ~2000 seeds / plate. The plates were then covered with plastic wrap and placed in a growth chamber. As the seedlings emerged from the soil, the plastic wrap was pierced to allow the humidity to gradually decrease. After two weeks, the plastic wrap was completely removed. The plates were moved to the greenhouse and fertilized with NPK fertilizer. The seedlings were reinserted into the floating trays and grown to transplant size. The plants were then transplanted into the field. During the growth process, the plants self-pollinated to form M1 seeds. At maturity, 5 seeds were harvested from each plant, and individual names were given to the seed groups from each plant. Thus, the M1 population was formed. A mixture of M1 seeds from each M0 plant was grown, and leaves of the M1 plants were collected for DNA extraction. Target genes in, around, or on both sides of the Nic1b region were amplified and sequenced for mutation identification. Mutations were identified in all 35 NCGs (NCG1-NCG35), with emphasis on all NCGs showing differential expression between TN90 and LA BU21, especially those encoding ERF-like proteins.

[0570] Example 6: Ta...

Claims

1. A modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having at least 90% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204 and 205.

2. The modified tobacco plant or part thereof of claim 1, wherein the polynucleotide has at least 95% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204, and 205.

3. The modified tobacco plant or part thereof of claim 1, wherein the polynucleotide has 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204, and 205.

4. The modified tobacco plant or part thereof of claim 1, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid as compared to a control tobacco plant lacking the mutation.

5. The modified tobacco plant or part thereof of claim 4, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

6. A modified tobacco plant or part thereof comprising a non-natural mutation in a polynucleotide having a nucleic acid sequence encoding a polypeptide having at least 90% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

7. The modified tobacco plant or part thereof of claim 6, wherein the polynucleotide has a nucleic acid sequence encoding a polypeptide having at least 95% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

8. The modified tobacco plant or part thereof of claim 6, wherein the polynucleotide has a nucleic acid sequence encoding a polypeptide having 100% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207.

9. The modified tobacco plant or part thereof of claim 6, wherein relative to a control plant not having the genetic modification or mutation or recombinant nucleic acid construct, the modified tobacco plant comprises a reduced level of at least one alkaloid compared to a control tobacco plant lacking the mutation.

10. The modified tobacco plant or part thereof of claim 6, wherein the higher USDA Grade Index value is at least 5% higher than the comparable leaves of the control plant.

11. The tobacco plant or part thereof of claim 9, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

12. Cured tobacco material derived from the tobacco plant of claim 6.

13. A tobacco product comprising the cured tobacco material of claim 12.

14. The tobacco product of claim 13, wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated groove filter cigarettes, ventilated groove filter cigarettes, cigars, snuff, pipe tobacco, cigarillos, cigarettes, chewing tobacco, leaf tobacco, cut and shredded tobacco, reconstituted tobacco, loose leaf chewing tobacco, plugged chewing tobacco, moist snuff, snuff and dry snuff or a smokeless tobacco product.

15. A method for producing a modified tobacco plant, comprising: (a) inducing a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 83, 84, 87-89, 180, 181, 206 and 207 in at least one tobacco cell; (b) selecting at least one tobacco cell from step (a) that comprises the non-natural mutation; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid as compared to a control tobacco plant lacking the mutation.

16. The method of claim 15, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation.

17. The method of claim 15, wherein the endogenous nucleic acid sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3, 13, 14, 17-19, 38, 48, 49, 52-54, 153, 154, 158, 159, 202, 203, 204, and 205.

18. The method of claim 15, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

19. The method of claim 15, wherein the method further comprises: (d) growing the modified tobacco plant regenerated in step (c).

20. The method of claim 19, wherein the method further comprises: (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; as well as (f) obtaining at least one seed from said crossing in step (e).

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